Implementation of an effective time-saving two-stage methodology for microstructural characterization of cemented carbides

Linear intercept on scanning electron microscopy micrographs is the most commonly used measurement method to determine carbide grain size and contiguity in WC–Co cemented carbides (hardmetals). However, it involves manual time-consuming measurements and is critically dependent on the quality of the micrographs as well as on the identification and definition of grain boundaries. In this study a two-stage methodology for microstructural characterization of hardmetals is presented. First, a digital semi-automatic image analysis procedure for grain size determination of the carbide phase is presented. It involves an experimental assessment of grain size on processed images corresponding to a series of WC–Co and WC–Ni cemented carbide grades with different microstructural characteristics. Obtained results are then compared to the values obtained by means of the linear intercept technique. A good correlation between the mean grain sizes determined following both measurement techniques was attained. Based on experimental findings, a series of empirical relations were found to correlate grain size distributions obtained following both methods. Second, an empirical relation for estimating carbide contiguity in WC–Co cemented carbides is proposed. This relation considers simultaneously the influence of the binder content and the experimentally determined mean grain size on contiguity. The proposed equation for contiguity estimation is based on extensive data collection from open literature. An excellent agreement was attained between contiguity values estimated from such equation and those obtained using the linear intercept technique. This validates the two-stage procedure as an effective time-saving methodology for microstructural characterization of WC–Co cemented carbides.Peer ReviewedPostprint (author's final draft

Cytomegalovirus drug resistance mutations in transplant recipients with suspected resistance

Resistant CMV infections are challenging complications after SOT and HSCT. Prompt recognition of ARMs is imperative for appropriate therapy. 108 plasma samples from 96 CMV + transplant recipients with suspected resistance were analysed in CNM in a retrospective nationwide study from January 2018 to July 2022 for resistance genotyping. ARMs in UL97 and UL54 were found in 26.87% (18/67) and 10.60% (7/66) of patients, respectively. Patients' ARM distribution in UL97 was as follows: L595S n = 3; L595S/M460I n = 1; L595S/N510S n = 1; L595W n = 1; C603W n = 4; A594V n = 3; A594E n = 1; C607Y n = 1; L397R/T409M/H411L/M460I n = 1; L397I n = 1; H520Q n = 1; four patients showed ARMs in UL54 as well (F412C n = 1; T503I n = 2; P522S n = 1), whereas three patients exhibited ARMs in UL54 only (L501I/T503I/L516R/A834P n = 1; A987G n = 2). L516R in UL54 and L397R/I and H411L in UL97 have been found for the first time in a clinical sample. L595S/W was the most prevalent ARM found to lend resistance to GCV. In UL54 all ARMs lent resistance to GCV and CDV. In addition, A834P, found in one patient, also lent resistance to FOS. CMV load did not differ significantly in patients with or without ARMs, and no differences were found either between patients with ARMs in UL97 or in UL97 and UL54. Despite extensive use of classical antivirals for the treatment of CMV infection after HSCT and SOT, ARMs occurred mainly in viral UL97 kinase, which suggests that CDV and mostly FOS continue to be useful alternatives to nucleoside analogues after genotypic detection of ARMs.This work was supported by a grant from Instituto de Salud Carlos III. AESI2021 PCIII00011-MPY434/2021. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.S

Molecular epidemiology of Kaposi sarcoma virus in Spain

Background: Since human herpesvirus 8 (HHV-8) infection may be underestimated and HHV-8 subtype circulation in Spain remains unknown, a molecular epidemiologic study is highly desirable. Objectives: This study aimed to analyse HHV-8 subtype diversity and their distribution in Spain. Study design: The study included 142 HHV-8 infected patients. A nested PCR was developed in order to permit Sanger sequencing of HHV-8 K1 ORF directly from clinical samples received at the CNM from 2013 to 2021. Phylogenetic characterization was performed. Results: Genotypes A and C comprised 55.6% and 42.3% of strains. Regarding subtypes, 25.4% of strains were C3, 19.7% were A3, 14.1% were A5, and C2, A1, A4, C1, A2, C7 were 11.3%, 11.3%, 8.5%, 4.2%, 2.1% and 1.4%, respectively. Subtype E1, E2 and B1 were found in only one patient each (0.7%). The Madrid region accounted for 52.1% of patients and showed a significantly different subtype distribution compared to the others (P = 0.018). Subtypes B1, E1, and E2 were observed to appear sporadically, although overall genotypes A and subtype C3 remained the most frequent and unwavering. Subtype A3 presented the highest diversity as displayed by the highest number of clusters in phylogenetic analysis. Non-significant differences in viral loads between genotypes were found, but significantly higher viral loads in subtype C2 compared to subtype C3 was found, while no significant subtype differences were observed between subtypes within genotype A. Infections with HHV-8 were detected in 94 (66.2%) patients without KS and compared to patients with KS non-significant differences in subtype distribution were found. Conclusions: Subtype prevalence and regional distribution followed a similar pattern compared to other western European countries. Our study is the first to report HHV-8 subtypes E1 and E2 circulating in Europe that might be reflective of migration of population from Caribbean countries. Our study suggests that infection by HHV-8 is underestimated, and wider screening should be recommended for risk groups.This work was supported by funds and a grant from Instituto de Salud Carlos III. Project code MPY 1372/2012 and MPY 434/2021. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Shotgun metagenomics to investigate unknown viral etiologies of pediatric meningoencephalitis

Introduction: Meningoencephalitis in children poses a diagnostic challenge, as etiology remains unknown for most of patients. Viral metagenomics by shotgun sequencing represents a powerful tool for investigating unknown viral infections related to these cases. Patients and methods: In a two-year, reference-centre, retrospective study, we investigated the usefulness of viral metagenomics of cerebrospinal fluid (CSF) for the diagnosis of viral infectious meningoencephalitis in forty seven pediatric patients, forty of them previously tested negative with a routine neurologic panel of viral targets that included herpesvirus 1-3 and enterovirus. We enhanced the detection by targeting viral sequences by hybrid capture. Raw sequence data was analysed using three bioinformatics pipelines. Results: Out of forty remaining children with meningoencephalitis of unknown viral etiology, a significant detection of viral nucleic acid by shotgun sequencing was found in twenty one, which was confirmed in ten of them by specific PCR: seven human endogenous retrovirus K113 (HER K113), one parechovirus 3, one human herpesvirus 5 (HHV5); one enterovirus B (Echovirus 9). The remaining eleven CSF were not confirmed by PCR: three rotavirus, one human herpesvirus 7 (HHV7), one influenza A, one mastadenovirus C, one sindbis virus, one torque teno virus, one human immunodeficiency virus 1 (HIV-1), one human alphaherpesvirus 3 (HHV3), one human alphaherpesvirus 2 (HHV2). Conclusions: Underutilization of currently available meningitis-encephalitis diagnostic techniques such as BioFire® FilmArray® is the main cause of undiagnosed cases of meningoencephalitis. However, in this study we detected uncommon viruses that should be considered, including virus, rotavirus, sindbis virus, influenza A virus and HHV7. No other viral sequences that could be readily linked to CNS inflammation were detected. Some findings may stem from reagent or sample contamination, as seen with papillomavirus; for others, the clinical relevance of the virus remains uncertain and should be substantiated by further studies, as is the case with endogenous retrovirus K113 virus. Online bioinformatics pipeline CZID represents a valuable tool for analysing shotgun sequencing data in cases of neurological conditions with unknown etiology. Altogether, this study highlights the potential of shotgun sequencing in identifying previously unknown viral neuropathogens and sheds light on the interpretation issues related to its application in clinical microbiology.This work was supported by a grant from Instituto de Salud Carlos III. Project code MPY434/2021. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Varicella-zoster virus clades circulating in Spain over two decades

BACKGROUND: Despite childhood universal VZV immunization was introduced in 2015, there are no data on VZV clade distribution in Spain. OBJECTIVES: To characterize the varicella-zoster virus strains circulating in Spain between 1997 and 2016. STUDY DESIGN: In this retrospective study, we determined the VZV clades in 294 patients with different pathologies (mainly encephalitis, zoster and varicella) by sequencing three fragments within ORF 22, ORF 21 and ORF 50 and, subsequently analyzing 7 relevant SNPs. RESULTS: Among these 294 patients, 132(44.9%) patients were infected by clade 1, 42(14.3%) patients by clade 3, 19(6.5%) by clade 5, 29(9.9%) by clade VI and 3(1%) by clade 4. Four patients (1.4%) were infected by clade 2 vOKA strains, who received one dose of live-attenuated varicella vaccine. Putative recombinant clade 1/3 was identified in 6 cases (2.0%). Results obtained from partial sequences were assigned to clade 1 or 3 in 56(19%) patients and clade 5 or VI in 3(1.0%) patients. In the multivariate analysis, encephalitis was independently associated with clades 1 and 3 and age >14y.o. (P = 0.035 and P = 0.021, respectively). Additionally, Madrid had significant fewer cases of encephalitis compared with the rest of regions analyzed (P = 0.001). CONCLUSIONS: Higher prevalence of clades 1 and 3 and their relation with encephalitis and age >14y.o. suggest earlier introduction of this clades in Spain. Putative interclade 1 and 3 recombinants are circulating in patients with encephalitis, herpes zoster and varicella. Several cases were related to vOKA vaccination but vaccine strains do not seem to circulate in the general population.This work was supported by a grant from Instituto de Salud Carlos III. Project code MPY1372/12. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Pediatric Parapneumonic Empyema, Spain

Increased incidence is principally due to highly invasive nonvaccine serotypes of pneumococci, especially serotype 1

Mutations in Coding and Non-Coding Regions in Varicella-Zoster Virus Causing Fatal Hemorrhagic Fever Without Rash in an Immunocompetent Patient: Case Report

Introduction: We report the case of a fatal hemorrhagic varicella primary infection in an immunocompetent man and whole-genome characterization of the virus for the investigation of biomarkers of virulence. Case: A 38-year-old patient born in Nigeria presented to the emergency department with abdominal pain and subsequently developed fatal hemorrhagic disease without skin rash. Extensive laboratory tests including serology and PCR for arenaviruses, bunyaviruses and ebolaviruses were negative. Varicella-zoster virus (VZV) PCR of sera, liver and spleen tissue samples from autopsy revealed the presence of VZV DNA. Primary infection by varicella-zoster virus with hemorrhagic manifestations was diagnosed after virological testing. The VZV genome was sequenced using a mWGS approach. Bioinformatic analysis showed 53 mutations across the genome, 33 of them producing non-synonymous variants affecting up to 14 genes. Some of them, such as ORF11 and ORF 62, encoded for essential functions related to skin or neurotropism. To our knowledge, the mutations reported here have never been described in a VZV causing such a devastating outcome. Discussion: In immunocompetent patients, viral factors should be considered in patients with uncommon symptoms or severe diseases. Some relevant mutations revealed by using whole genome sequencing (WGS) directly from clinical samples may be involved in this case and deserves further investigation. Conclusion: Differential diagnosis of varicella-zoster virus in immunocompetent adults should be considered among patients with suspected VHF, even if the expected vesicular rash is not present at admission and does not arise thereafter. Whole genome sequencing of strains causing uncommon symptoms and/or mortality is needed for epidemiological surveillance and further characterization of putative markers of virulence. Additionally, this report highlights the recommendation for a VZV vaccination policy in non-immunized migrants from developing countries.This work was supported by a grant from Instituto de Salud Carlos III. Project code MPY1372/12. The journal’s Rapid Service fee was paid by Consorcio Centro de Investigación en Red (CIBER). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Cytomegalovirus resistance in transplant patients Review

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