Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

[Providing studies quality for pesticides risk evaluation in their use according to proper laboratory practice rules].

The article covers experience of proper laboratory practice in hygienic studies examining air and workers' skin for assessment of exposure levels of pesticides in natural conditions of agricultural production.

Does proficiency testing improve the quality of hantavirus serodiagnostics? Experiences with INSTAND EQA schemes.

Hantavirus infections in Germany appear periodically with peak numbers every 2-3 years. The reported cases in the years 2007, 2010 and 2012 exceeded many times over those in the years in-between. In order to reveal faults of certain in vitro diagnostic assays (IVDs), to harmonize the performances of the individual assays and to improve the users' competence in interpreting the results, the National Consiliary Laboratory for Hantaviruses and INSTAND e.V. (Society for Promoting Quality Assurance in Medical Laboratories e.V.) established an external quality assessment (EQA) scheme for proficiency testing of hantavirus serodiagnostics. The first EQA scheme (pilot study) started in March 2009 with 58 participating laboratories from Germany and neighboring countries. Twice a year four serum samples were sent out to the participants to investigate whether the sample reflects an acute or past infection and to distinguish between infections with the hantavirus types Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV), both endemic in Central Europe. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied in the participating laboratories. An increasing number of laboratories participated, with a maximum of 92 in March 2014. When summarizing in total 2592 test results, the laboratories reached an overall specificity of 96.7% and a sensitivity of 95% in their detection of a hantavirus infection. A correct distinction between acute and past infections was forwarded in 90-96% of replies of laboratories. Exact serotyping (PUUV vs. DOBV) of the infection was reported in 81-96% of replies with the lowest accuracy for past DOBV infections; cross-reactivities between diagnostic antigens of the two viruses as well as persistent IgM titers in humans may interfere with exact testing. The EQAs revealed acceptable results for the serodiagnostic of hantavirus infection including serotyping but further improvement is still needed.

[Analysis of the results of the SEIMC External Quality Control Program. Year 2013]. / Análisis de resultados del Programa Externo de Control de Calidad SEIMC. Año 2013.

The External Quality Control Program of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) include controls for bacteriology, serology, mycology, parasitology, mycobacteria, virology, molecular microbiology and HIV-1, HCV and HBV viral loads. This manuscript presents the analysis of results obtained of the participants from the 2013 SEIMC External Quality Control Programme, except viral loads controls, that they are summarized in a manuscript abroad. As a whole, the results obtained in 2013 confirm the excellent skill and good technical standards found in previous editions. However, erroneous results can be obtained in any laboratory and in clinically relevant determinations. Once again, the results of this program highlighted the need to implement both internal and external controls in order to assure the maximal quality of the microbiological tests.

Challenges for accurate susceptibility testing, detection and interpretation of ß-lactam resistance phenotypes in Pseudomonas aeruginosa: results from a Spanish multicentre study.

OBJECTIVES: To evaluate the proficiency of Spanish laboratories regarding accurate susceptibility testing, detection and interpretation of Pseudomonas aeruginosa ß-lactam resistance phenotypes. METHODS: Thirteen characterized strains were sent to 54 participating centres: clinical strains producing horizontally acquired ß-lactamases [extended-spectrum ß-lactamases (ESBLs; PER-1 and OXA-161) and class A (GES-5) and B (VIM-2) carbapenemases] and mutants with combinations of chromosomal mechanisms (AmpC, OprD and/or efflux). The centres were requested to evaluate six antipseudomonal ß-lactams, provide raw/interpreted clinical categories and detect/infer the resistance mechanisms. Consensus results from reference centres were used to assign minor, major or very major errors (mEs, MEs or VMEs). RESULTS: Vitek2, MicroScan WalkAway and Wider were the most used devices (25%-30% each). CLSI/EUCAST breakpoints were used in 86%/14% of the determinations. Discrepancies exclusively due to the differential application of breakpoints were highest for aztreonam, followed by piperacillin/tazobactam. The lowest percentage of VMEs was for Vitek2, followed by Wider. The highest percentages of VMEs (6%) were for the AmpC-hyperproducing OprD(-) strain and for the GES-5 producer, while among antibiotics the highest percentage of VMEs (22%) involved piperacillin/tazobactam. Appropriate inference of resistance mechanisms was high for the VIM-2-producing strain (83%), but low (<40%) for strains producing ESBLs or non-metallo-ß-lactamase carbapenemases. CONCLUSIONS: The use of different breakpoints and devices, the complexity of mutation-driven resistance mechanisms and the lack of unequivocal tests to detect ESBLs or carbapenemases in P. aeruginosa leads to extraordinary variability and low accuracy in susceptibility testing, which may have consequences for the treatment and control of nosocomial infections.

External quality assessment of pertussis serology in Germany.

The purpose of this investigation was to test the performance of pertussis serology in diagnostic laboratories. The World Health Organization (WHO) Reference Reagent (06/142) and a sample with a low level of antibodies were sent to 200 participants of an external quality assessment (EQA) programme in Germany. The results were reported qualitatively and quantitatively, and were converted into IU/ml when possible. A total of 183 participants reported results. IgG, IgA and IgM enzyme-linked immunosorbent assays (ELISAs) with mixed antigens were used by 111, 110 and 113 participants, respectively, and 69 and 44 participants used IgG and IgA ELISAs with purified pertussis toxin (PT), respectively. IgG, IgA and IgM immunoblots were employed by 62, 63 and 11 participants, respectively. Most tests could distinguish between the positive and negative samples, but quantitative results were reported partly in non-comparable units. Only 37 % of participants used ELISAs that gave results comparable to the expected values in IU/ml and that could be interpreted according to published recommendations.

[On the quality control in forensic biochemical departments].

The main principles of quality control in the laboratory practice of forensic biochemical departments are discussed. Recommendations on international standardization are suggested. Characteristics of the main phases of intralaboratory quality control of investigations and the reagents used in them are described. The necessity of quality control at all stages of forensic-biochemical studies and expertise is emphasized.

First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides: A pilot study.

BACKGROUND: Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used. METHODS AND PRINCIPAL FINDINGS: A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing. CONCLUSIONS/SIGNIFICANCE: We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.

[Theoretic and practical contribution of bayesian inference to statistical process control: The experience of the Lyon Hospitals Board hemostasis laboratory]. / Intérêts théoriques et pratiques de l'inférence bayésienne dans la maîtrise statistique des procédés : retours d'expérience du laboratoire d'hémostase aux Hospices civils de Lyon.

Laboratories need to set up effective overall management of their internal quality control (IQC) and external quality assessment (EQA) results as key elements in statistical process control. Quality targets need to be defined, with methods to ensure durable control with respect to the relevant specifications. The hemostasis laboratory of the Lyon Hospitals Board (HCL, Lyon, France) uses model 3 from the Milan consensus conference, which is the state of the art in terms of quality targets, and uses a common EQA provider supplying as many real patient samples as possible. Giving priority to adopted methods, the lab optimizes the use of manufacturers' prior data: maximum acceptable inter assay coefficient of variation (CV) and prior IQC target values. Bayesian inference brings the method under control with respect to the manufacturers' prior data without the need for a preliminary phase. It links the IQC and EQA plans by the maximum acceptable CVs defined by the manufacturer.

First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.

BACKGROUND: Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. METHODS: For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. RESULTS AND CONCLUSION: A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.

Time to Get Serious about Measurement in Synthetic Biology.

For synthetic biology to mature, composition of devices into functional systems must become routine. This requires widespread adoption of comparable and replicable units of measurement. Interlaboratory studies organized through the International Genetically Engineered Machine (iGEM) competition show that fluorescence can be calibrated with simple, low-cost protocols, so fluorescence should no longer be published without units.

Towards an evaluation framework for Laboratory Information Systems.

INTRODUCTION: Laboratory testing and reporting are error-prone and redundant due to repeated, unnecessary requests and delayed or missed reactions to laboratory reports. Occurring errors may negatively affect the patient treatment process and clinical decision making. Evaluation on laboratory testing and Laboratory Information System (LIS) may explain the root cause to improve the testing process and enhance LIS in supporting the process. This paper discusses a new evaluation framework for LIS that encompasses the laboratory testing cycle and the socio-technical part of LIS. METHODOLOGY: Literature review on discourses, dimensions and evaluation methods of laboratory testing and LIS. A critical appraisal of the Total Testing Process (TTP) and the human, organization, technology-fit factors (HOT-fit) evaluation frameworks was undertaken in order to identify error incident, its contributing factors and preventive action pertinent to laboratory testing process and LIS. RESULT: A new evaluation framework for LIS using a comprehensive and socio-technical approach is outlined. Positive relationship between laboratory and clinical staff resulted in a smooth laboratory testing process, reduced errors and increased process efficiency whilst effective use of LIS streamlined the testing processes. CONCLUSION: The TTP-LIS framework could serve as an assessment as well as a problem-solving tool for the laboratory testing process and system.

Japanese external quality assessment program to standardize HIV-1 drug-resistance testing (JEQS2010 program) using in vitro transcribed RNA as reference material.

To design appropriate antiretroviral therapy regimens and avoid the emergence of human immunodeficiency virus (HIV)-1 variants with reduced susceptibility to antiretroviral drugs, genotypic drug-resistance testing (HIV genotyping) is strongly recommended. To monitor the quality of HIV genotyping in Japan, we performed an external quality assessment (EQA), named the Japanese external quality assessment program, to standardize HIV genotyping (JEQS). To accurately evaluate the quality of HIV genotyping, we employed as reference material (RM) a well-characterized sample, in vitro transcribed RNA (trRNA) that includes the HIV gag-pol sequence, and created a JEQS2010 panel consisting of three single variant and three mixed trRNA samples. All 11 participating laboratories showed high concordance rates (>96%) for the single variant samples. Eight laboratories also showed good rates of detecting minor variants, but three laboratories failed to detect the variants comprising one-half of the sample. These three laboratories used a common primer that had four internal mismatches to the minor trRNA clone. This program showed the usefulness of trRNA as RM, the high quality of HIV genotyping, and extensive interlaboratory variation in the ability to detect minor variants. These results suggest that improving the quality of HIV genotyping in Japan requires regularly implementing the EQA program and improving the HIV genotyping protocol in each laboratory.

Role of secondary level laboratories in strengthening quality at primary level health facilities' laboratories: an innovative approach to ensure accurate HIV, tuberculosis, and malaria test results in resource-limited settings.

Providing regular external quality assessment of primary level laboratories and timely feedback is crucial to ensure the reliability of testing capacity of the whole laboratory network. This study was aimed to assess the diagnostic performances of primary level laboratories in Southwest Showa Zone in Ethiopia. An external quality assessment protocol was devised whereby from among all the samples collected on-site at 4 health centers (HCs), each HC sent to a district hospital (DH) on a weekly basis 2 TB slides (1 Ziehl-Neelsen stained and another unstained), 2 malaria slides (1 Giemsa stained and another unstained), and 2 blood samples for HIV testing (1 whole blood and another plasma) for a comparative analysis. Similarly, the DH preserved the same amount and type of specimens to send to each HC for retesting. From October to November 2011, 192 single-blinded specimens were rechecked: 64 TB slides, 64 malaria slides, and 64 blood specimens for HIV testing. The analyses demonstrated an overall agreement of 95.3% (183/192) between the test and the retest, and 98.4% (63/64), 92.2% (59/64,) and 95.3% (61/64) for TB microscopy, malaria microscopy, and HIV rapid testing, respectively. Of the total TB slides tested positive, 20/23 (87%) were quantified similar in both laboratories. The agreement on HIV rapid testing was 100% (32/32) when plasma samples were tested either at HC (16/16) or at DH (16/16), while when whole blood specimens were tested, the agreement was 87.5% (14/16) and 93.8% (15/16) for samples prepared by HCs and DH, respectively. Results of this new approach proved that secondary laboratories could play a vital role in assuring laboratory qualities at primary level HCs, without depending on remotely located national and regional laboratories to provide this support.

State of affairs of tuberculosis in prison facilities: a systematic review of screening practices and recommendations for best TB control.

BACKGROUND: Prisoners are at high risk of developing tuberculosis (TB), causing morbidity and mortality. Prison facilities encounter many challenges in TB screening procedures and TB control. This review explores screening practices for detection of TB and describes limitations of TB control in prison facilities worldwide. METHODS: A systematic search of online databases (e.g., PubMed and Embase) and conference abstracts was carried out. Research papers describing screening and diagnostic practices among prisoners were included. A total of 52 articles met the inclusion criteria. A meta-analysis of TB prevalence in prison facilities by screening and diagnostic tools was performed. RESULTS: The most common screening tool was symptom questionnaires (63·5%), mostly reporting presence of cough. Microscopy of sputum with Ziehl-Neelsen staining and solid culture were the most frequently combined diagnostic methods (21·2%). Chest X-ray and tuberculin skin tests were used by 73·1% and 50%, respectively, as either a screening and/or diagnostic tool. Median TB prevalence among prisoners of all included studies was 1,913 cases of TB per 100,000 prisoners (interquartile range [IQR]: 332-3,517). The overall annual median TB incidence was 7·0 cases per 1000 person-years (IQR: 2·7-30·0). Major limitations for successful TB control were inaccuracy of diagnostic algorithms and the lack of adequate laboratory facilities reported by 61·5% of studies. The most frequent recommendation for improving TB control and case detection was to increase screening frequency (73·1%). DISCUSSION: TB screening algorithms differ by income area and should be adapted to local contexts. In order to control TB, prison facilities must improve laboratory capacity and frequent use of effective screening and diagnostic tools. Sustainable political will and funding are critical to achieve this.

Proficiency panel testing--a reliable tool in external quality assessment of sputum smear microscopy services in Gujarat, India.

OBJECTIVE: To assess the proficiency of Senior TB Laboratory Supervisors (STLSs) and district level Laboratory Technicians (LTs) in sputum smear microscopy. METHOD: Intermediate Reference Laboratory (IRL), Ahmedabad had manufactured and validated Proficiency Panel Testing slides from sputum samples, made On Site Evaluation (OSE) visits of District TB Centres (DTCs) in two rounds, and conducted Proficiency Panel Testing of STLSs & DTC-LTs from January 2005 to June 2009. RESULTS: High level of concordance in Z-N smear grading was found between Microbiologist and district laboratory staff. DTC readers reported overall consistency level of more than 98% in Z-N grade agreement during both the IRL, EQA, OSE visits. The tendency to over-grade the panel slides was much higher (more than 22%) as compared to under-grade (less than 2%) them in "correct slides". High False Positive (HFP) error was not observed in the present study. CONCLUSION: Laboratory supervisor's proficiency can be quickly assessed by Proficiency Panel Testing, under multi-level quality assurance network system of sputum smear microscopy in public health programmes like the RNTCP. Proficiency Panel Testing is highly replicable and reproducible tool for quick and reliable assessment of proficiency of the staff and it can be made more effective by raising the proportion of lower grade positive slides in panel set of each reader. DTC readers' overall agreement level of more than 98% in Z-N grade suggests high level of precision and excellent consistency during both the IRL, EQA, OSE rounds. It is concluded that even for a large network of sputum smear microscopy centres under public health programmes like the RNTCP in order to take corrective action, Proficiency Panel Testing can be effectively used for quick identification of suboptimal- technical performance of the supervisory staff.