UNLABELLED: The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 μL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used (P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE: Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.

• MeSH
• Molecular Diagnostic Techniques standards   methods   MeSH
• DNA, Fungal * blood   genetics   MeSH
• Real-Time Polymerase Chain Reaction * standards   methods   MeSH
• Humans MeSH
• Mucorales * genetics   isolation & purification   MeSH
• Mucormycosis * diagnosis   microbiology   blood   MeSH
• Sensitivity and Specificity * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH

Syphilis is a multistage sexually transmitted disease caused by Treponema pallidum ssp. pallidum (TPA). This study analyzed clinical samples collected from patients with a diagnosed syphilis infection from 2004-2022, isolated in the Czech Republic. Mucocutaneous swab samples (n = 543) from 543 patients were analyzed, and from these samples, 80.11 % (n = 435) were PCR positive, and 19.89 % (n = 108) were PCR negative for TPA DNA. Swabs were more often positive when collected from syphilis patients in the primary and secondary stages, compared to the latent or unknown stage. There was no significant difference in PCR positivity between the primary and secondary stages (p = 0.099). In IgM-positive patients, a statistically significant association with PCR-positivity was found in samples from seropositive (p = 0.033) and serodiscrepant (RPR negative) patients (p = 0.0006). When assessing our laboratory-defined cases of syphilis, the RPR, IgM, and PCR tests were similarly effective (within the range of 80.1-86.1 %). However, parallel testing with these methods was even more effective, i.e., RPR + PCR was 96.1 % effective and RPR + IgM + PCR was 97.8 % effective. A combination of RPR + PCR, or a combination of all three tests (RPR, IgM, and PCR) can therefore be used to reliably detect active syphilis cases, including reinfections. Our findings show that the reverse algorithm for detecting syphilis could be substantially improved by adding IgM and PCR testing.

• MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Immunoglobulin M * blood   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Polymerase Chain Reaction * methods   MeSH
• Antibodies, Bacterial blood   MeSH
• Sensitivity and Specificity MeSH
• Syphilis Serodiagnosis methods   MeSH
• Syphilis * diagnosis   microbiology   MeSH
• Treponema pallidum * genetics   isolation & purification   immunology   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main causes of economic losses in sericulture. Thus, it is essential to establish rapid and effective method for BmNPV detection. In the present study, we have developed a recombinase-aided amplification (RAA) to amplify the BmNPV genomic DNA at 37 °C within 30 min, and achieved a rapid detection method by coupling with a lateral flow dipstick (LFD). The RAA-LFD method had a satisfactory detection limit of 6 copies/μL of recombinant plasmid pMD19-T-IE1, and BmNPV infection of silkworm can be detected 12 h post-infection. This method was highly specific for BmNPV, and without cross-reactivity to other silkworm pathogens. In contrast to conventional polymerase chain reaction (PCR), the RAA-LFD assay showed higher sensitivity, cost-saving, and especially is apt to on-site detection of BmNPV infection in the sericulture production.

• MeSH
• Bombyx * virology   MeSH
• DNA, Viral genetics   MeSH
• Limit of Detection MeSH
• Nucleopolyhedroviruses * genetics   isolation & purification   MeSH
• Recombinases * metabolism   genetics   MeSH
• Sensitivity and Specificity MeSH
• Nucleic Acid Amplification Techniques * methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH

Remdesivir therapy has been declared as efficient in the early stages of Covid-19. Of the 339 patients (males 55.8%, age 71(59;77) years) with a detectable viral load, 140 were treated with remdesivir (of those 103 in the ICU and 57 immunosuppressed) and retrospectively compared with 199 patients (of those 82 in the ICU and 28 immunosuppressed) who were denied therapy due to advanced Covid-19. The viral load was estimated by detecting nucleocapsid antigen in serum (n = 155, median 217(28;1524)pg/ml), antigen in sputum (n = 18, COI 18(4.6;32)), nasopharyngeal antigen (n = 44, COI 17(8;35)) and the real-time PCR (n = 122, Ct 21(18;27)). After adjustment for confounders, patients on remdesivir had better 12-month survival (HR 0.66 (0.44;0.98), p = 0.039), particularly when admitted to the ICU (HR 0.49 (0.29;0.81), p = 0.006). For the immunocompromised patients, the difference did not reach statistical significance (HR 0.55 (0.18;1.69), p = 0.3). The other most significant confounders were age, ICU admission, mechanical ventilation, leukocyte/lymphocyte ratio, admission creatinine and immunosuppression. The impact of monoclonal antibodies or previous vaccinations was not significant. Despite frequent immune suppression including haemato-oncology diseases, lymphopenia, and higher inflammatory markers in the remdesivir group, the results support remdesivir administration with respect to widely available estimates of viral load in patients with high illness severity.

• MeSH
• Adenosine Monophosphate * analogs & derivatives   therapeutic use   MeSH
• Alanine * analogs & derivatives   therapeutic use   MeSH
• Antiviral Agents * therapeutic use   MeSH
• COVID-19 * virology   mortality   MeSH
• COVID-19 Drug Treatment * MeSH
• Intensive Care Units MeSH
• Middle Aged MeSH
• Humans MeSH
• Critical Care MeSH
• Retrospective Studies MeSH
• SARS-CoV-2 * drug effects   isolation & purification   physiology   MeSH
• Aged MeSH
• Severity of Illness Index MeSH
• Viral Load * drug effects   MeSH
• Treatment Outcome MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The current study assessed the performance of the fully automated RT-PCR-based IdyllaTM GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK, ROS1, RET, and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The IdyllaTM GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK, 33 ROS1, 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the IdyllaTM GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK, 96.7%/99.0% for ROS1, 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The IdyllaTM GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK, ROS1, RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.

• MeSH
• Anaplastic Lymphoma Kinase * genetics   MeSH
• Exons * genetics   MeSH
• Oncogene Proteins, Fusion * genetics   MeSH
• Gene Rearrangement MeSH
• In Situ Hybridization, Fluorescence methods   MeSH
• Humans MeSH
• Multiplex Polymerase Chain Reaction MeSH
• Biomarkers, Tumor genetics   analysis   MeSH
• Lung Neoplasms * genetics   pathology   MeSH
• Carcinoma, Non-Small-Cell Lung * genetics   pathology   MeSH
• Proto-Oncogene Proteins c-met * genetics   MeSH
• Proto-Oncogene Proteins c-ret * genetics   MeSH
• Proto-Oncogene Proteins * genetics   MeSH
• Protein-Tyrosine Kinases * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Multicenter Study MeSH

Portable household air purifiers are widely used devices designed to maintain a high-quality level of indoor air. Portable air purifiers equipped with the high-efficiency air (HEPA) filter served 100 h in a household space occupied by two adults without any symptoms of respiratory tract infection. The main objective of the study was to determine microbial contamination on the HEPA filter and to investigate if the selected nanotextile monolayer made of polyamide 6 (PA6) nanofibers can capture potential microorganisms when installed downstream of the HEPA filter as the final filtration medium. Samples were taken from the inlet and outlet surfaces. Samples from the nanotextile were collected in the same manner as from the HEPA filter. QIAStat DX® 1.0 Analyzer using the Respiratory SARS CoV-2 Panel multiplex PCR detection system was selected for microorganism detection. Adenovirus was detected on the inlet surface of the HEPA filter. The outlet surface of the filter contained no viruses included in the Respiratory SARS CoV-2 Panel portfolio. The nanotextile monolayer was replaced twice during the 100 h of operation, so three pieces were used and all contained coronavirus 229 E. Coronavirus 229 E was then detected in the nasopharynx of one of the members of the household as well. It may be assumed that the selected nanotextile is capable of capturing a virus of a small size.

• MeSH
• Filtration MeSH
• Humans MeSH
• Pilot Projects MeSH
• SARS-CoV-2 MeSH
• Severe Acute Respiratory Syndrome * MeSH
• Air Filters * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Global impact of COVID-19 pandemic has heightened the urgency for efficient virus detection and identification of variants such as the Q57H mutation. Early and efficient detection of SARS-CoV-2 among densely populated developing countries is paramount objective. Although RT-PCR assays offer accuracy, however, dependence on expansive kits and availability of allied health resources pose an immense challenge for developing countries. In the current study, RT-LAMP based detection of SARS-Cov-2 with subsequent confirmation of Q57H variant through ARMS-PCR was performed. Among the 212 collected samples, 134 yielded positive results, while 78 tested negative using RT-LAMP. Oropharyngeal swabs of suspected individuals were collected and processed for viral RNA isolation. Isolated viral RNA was processed further by using either commercially available WarmStart Master Mix or our in house developed LAMP master mix separately. Subsequently, the end results of each specimen were evaluated by colorimetry. For LAMP assays, primers targeting three genes (ORF1ab, N and S) were designed using PrimerExplorer software. Interestingly, pooling of these three genes in single reaction tube increased sensitivity (95.5%) and specificity (93.5%) of LAMP assay. SARS-CoV-2 positive specimens were screened further for Q57H mutation using ARMS-PCR. Based on amplicon size variation, later confirmed by sequencing, our data showed 18.5% samples positive for Q57H mutation. Hence, these findings strongly advocate use of RT-LAMP-based assay for SARS-CoV-2 screening within suspected general population. Furthermore, ARMS-PCR also provides an efficient mean to detect prevalent mutations against SARS-Cov-2.

• MeSH
• COVID-19 * diagnosis   MeSH
• Molecular Diagnostic Techniques methods   MeSH
• Humans MeSH
• Pandemics MeSH
• Polymerase Chain Reaction MeSH
• RNA, Viral genetics   MeSH
• SARS-CoV-2 * genetics   MeSH
• Sensitivity and Specificity MeSH
• COVID-19 Testing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Probiotic supplementation in childhood serves as an additional source of bacterial colonisers and represents an opportunity to beneficially manipulate the intestinal microbiome. Differences in the ability of probiotic strains to colonise the gut may be related to the variously diversified gut microbiome. We report the results of the association between composition of the gut microbiome and the colonisation capacity of the probiotic strain Escherichia coli A0 34/86 (CNB - Colinfant New Born supplement) in the cases of three healthy children in different development stages (infant, toddler, and pre-school), as a preliminary insight to possible future prospective studies of this subject. Microbiome composition was estimated by 16S rRNA gene sequencing of 55 stool samples collected during approximately 3.5-13 months long periods. Detailed characterisation of the E. coli population was performed using colony PCR to detect 33 E. coli genetic determinants. In all children, genetic determinants typical for the probiotic E. coli A0 34/86 strain were detected immediately after administration of the probiotics. Analysis of the initial sample composition (the last sample taken before the probiotic administration) showed that the gut microbiome of infant and toddler with lower bacterial diversity was more successfully colonised by the probiotic strain. In our case report of three children, we showed for the first that supplementation with CNB probiotics in early infancy and toddlerhood was associated with high E. coli A0 34/86 colonisation and a significant change in the composition of the gut microbiome. Our results indicate that administration of CNB for its recommended duration might be efficient only in very early childhood.

• MeSH
• Escherichia coli genetics   MeSH
• Infant MeSH
• Humans MeSH
• Microbiota * MeSH
• Child, Preschool MeSH
• Probiotics * MeSH
• Prospective Studies MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Check Tag
• Infant MeSH
• Humans MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH

"Genetics and Genomics in Medicine is a new textbook written for undergraduate and graduate students, as well as medical researchers, which explains the science behind the uses of genetics and genomics in medicine today. It is not just about rare inherited and chromosomal disorders, but how genetics affects the whole spectrum of human health and disease. DNA technologies are explained, with emphasis on the modern techniques that have revolutionized the use of genetic information in medicine and are indicating the role of genetics in common complex diseases. The detailed, integrative coverage of genetic approaches to treatment and prevention includes pharmacogenomics and the prospects for personalized medicine. Cancers are essentially genetic diseases and are given a dedicated chapter that includes new insights from cancer genome sequencing. Clinical disorders are covered throughout and there are extensive end-of-chapter questions and problems"--Provided by publisher.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

• MeSH
• Fusion Proteins, bcr-abl * genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * diagnosis   drug therapy   genetics   MeSH
• Imatinib Mesylate MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Neoplasm, Residual genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH