Monkeypox virus genomic accordion strategies.

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

Detecting, Quantifying, and Isolating Monkeypox Virus in Suspected Cases, Spain.

When a monkeypox virus outbreak began in several parts of the world in May 2022, timely and accurate diagnosis became mandatory. In our laboratory, a real-time quantitative PCR was designed and evaluated in several patient samples and compared with isolation results. Genomic viral load was related to virus viability.

Emergence of New SARS-CoV2 Omicron Variants after the Change of Surveillance and Control Strategy.

In January 2022, there was a global and rapid surge of the Omicron variant of SARS-CoV-2 related to more transmission. This coincided with an increase in the incidence in Asturias, a region where rapid diagnosis and containment measures had limited the circulation of variants. METHODS: From January to June 2022, 34,591 variants were determined by the SNP method. From them, 445 were characterized by the WGS method and classified following pangolin program and phylogenic analysis. RESULTS: The Omicron variant went from being detected in 2438 (78%) samples in the first week of January 2021 to 4074 (98%) in the third week, according to the SNP method. Using the WGS method, 159 BA.1 (35.7%), 256 BA.2 (57.6%), 1 BA.4 (0.2%) and 10 BA.5 (2.2%) Omicron variants were found. Phylogenetic analysis detected that three new sub-clades, BA.2,3.5, BA.2.56 and BF1, were circulating. CONCLUSIONS: The increase in the incidence of SARS-CoV2 caused the circulation of new emerging variants. Viral evolution calls for continuous genomic surveillance.

Enhanced Detection of Viral RNA Species Using FokI-Assisted Digestion of DNA Duplexes and DNA/RNA Hybrids.

The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.

FURIN gene variants (rs6224/rs4702) as potential markers of death and cardiovascular traits in severe COVID-19.

Furin is a protease that plays a key role in the infection cycle of SARS-CoV-2 by cleaving the viral proteins during the virus particle assembly. In addition, Furin regulates several physiological processes related to cardio-metabolic traits. DNA variants in the FURIN gene are candidates to regulate the risk of developing these traits as well as the susceptibility to severe COVID-19. We genotyped two functional FURIN variants (rs6224/rs4702) in 428 COVID-19 patients in the intensive care unit. The association with death (N = 106) and hypertension, diabetes, and hyperlipidaemia was statistically evaluated. The risk of death was associated with age, hypertension, and hypercholesterolemia. The two FURIN alleles linked to higher expression (rs6224 T and rs4702 A) were significantly increased in the death cases (odds ratio= 1.40 and 1.43). Homozygosis for the two high expression genotypes (rs6224 TT and rs4702 AA) and for the T-A haplotype was associated with an increased risk of hypercholesterolemia. In the multiple logistic regression both, hypercholesterolemia and the TT + AA genotype were significantly associated with death. In conclusion, besides its association with hypercholesterolemia, FURIN variants might be independent risk factors for the risk of death among COVID-19 patients.

Association between the interferon-induced transmembrane protein 3 gene (IFITM3) rs34481144 / rs12252 haplotypes and COVID-19.

The interferon induced transmembrane-protein 3 (IFITM3) plays an important role in the defence against viral infection. IFITM3 gene variants have been linked to differences in expression and associated with the risk of severe influenza by some authors. More recently, these variants have been associated with the risk of COVID-19 after SARS-CoV-2 infection. We determined the effect of two common IFITM3 polymorphisms (rs34481144 â€‹C/T and rs12252 A/G) on the risk of hospitalization due to COVID-19 by comparing 484 patients (152 required support in thr intensive care unit, ICU) and 182 age and sex matched controls (no disease symptoms). We found significantly higher frequencies of rs34481144 â€‹T and rs12252 â€‹G carriers among the patients (OR â€‹= â€‹2.02 and OR â€‹= â€‹1.51, respectively). None of the two variants were associated with ICU-admission or death. We found a significantly higher frequency of rs34481144 CC â€‹+ â€‹rs12252 AA genotype carriers among the controls, suggesting a protective effect (p = 0.001, OR = 0.56, 95%CI = 0.40-0.80). Moreover, haplotype rs34481144 â€‹C - rs12252 A was significantly increased in the controls (p â€‹= â€‹0.008, OR â€‹= â€‹0.71, 95%CI â€‹= â€‹0.55-0.91). Our results showed a significant effect of the IFITM3 variants in the risk for hospitalization after SARS-CoV-2 infection.

Alterations in biochemical markers in adenovirus infection.

BACKGROUND: Analyze possible relationships between HAdV and markers for inflammation, specifically the C-reactive protein (CRP) and procalcitonin (PCT) tests, along with other haematological markers. METHODS: Retrospective study of 487 children presenting with fever and/or acute respiratory symptoms in the Paediatric Emergency Department. Analyses included viral presence/absence (both HAdV and other respiratory viruses) in respiratory exudates, CRP and PCT alterations in plasma, and haematological markers in whole blood. RESULTS: Viral load was >500 copies/103 cells of HAdV in 127 cases (26.1%), of which 66 (52%, P<0.0001) had alterations in PCT, and 112 (88.1%, P<0.0001) in CRP. Haematological markers were similar either HAdV was present or not, although many HAdV positive patients demonstrated leukocytosis (66%). Bacterial cultures from 141 samples showed altered PCT in 27 (60%) with HAdV infection, in 3 (18.7%) with bacterial infection, and 13 (26.5%) without either viral or bacterial infection (P<0.05). CRP was altered in 88.9% of HAdV infected children and in 87% infected with bacteria, although the percentage was greater than in cases where other respiratory viruses were present (61.3% P<0.05). CONCLUSIONS: Results demonstrate a clear relationship between HAdV infection and alterations in PCT and CRP which should be taken into account in paediatric patient management.

The Interferon-induced transmembrane protein 3 gene (IFITM3) rs12252 C variant is associated with COVID-19.

BACKGROUND AND AIMS: The interferon-induced transmembrane proteins play an important antiviral role by preventing viruses from traversing the cellular lipid bilayer. IFITM3 gene variants have been associated with the clinical response to influenza and other viruses. Our aim was to determine whether the IFITM3 rs12252 polymorphism was associated with the risk of developing severe symptoms of COVID-19 in our population. METHODS: A total of 288 COVID-19 patients who required hospitalization (81 in the intensive care unit) and 440 age matched controls were genotyped with a Taqman assay. Linear regression models were used to compare allele and genotype frequencies between the groups, correcting for age and sex. RESULTS: Carriers of the minor allele frequency (rs12252 C) were significantly more frequent in the patients compared to controls after correcting by age and sex (p = 0.01, OR = 2.02, 95%CI = 1.19-3.42). This genotype was non-significantly more common among patients who required ICU. CONCLUSIONS: The IFITM3 rs12252 C allele was a risk factor for COVID-19 hospitalization in our Caucasian population. The extent of the association was lower than the reported among Chinese, a population with a much higher frequency of the risk allele.

Angiotensin-converting enzymes (ACE, ACE2) gene variants and COVID-19 outcome.

The Angiotensin system is implicated in the pathogenesis of COVID-19. First, ACE2 is the cellular receptor for SARS-CoV-2, and expression of the ACE2 gene could regulate the individuals susceptibility to infection. In addition, the balance between ACE1 and ACE2 activity has been implicated in the pathogenesis of respiratory diseases and could play a role in the severity of COVID-19. Functional ACE1/ACE2 gene polymorphisms have been associated with the risk of cardiovascular and pulmonary diseases, and could thus also contribute to the outcome of COVID-19. We studied 204 COVID-19 patients (137 non-severe and 67 severe-ICU cases) and 536 age-matched controls. The ACE1 insertion/deletion and ACE2 rs2285666 polymorphism were determined. Variables frequencies were compared between the groups by logistic regression. We also sequenced the ACE2 coding nucleotides in a group of patients. Severe COVID-19 was associated with hypertension male gender (p < 0.001), hypertension (p = 0.006), hypercholesterolaemia (p = 0.046), and the ACE1-DD genotype (p = 0.049). In the multiple logistic regression hypertension (p = 0.02, OR = 2.26, 95%CI = 1.12-4.63) and male gender (p = 0.002; OR = 3.15, 95%CI = 1.56-6.66) remained as independent significant predictors of severity. The ACE2 polymorphism was not associated with the disease outcome. The ACE2 sequencing showed no coding sequence variants that could explain an increased risk of developing COVID-19. In conclusion, an adverse outcome of COVID-19 was associated with male gender, hypertension, hypercholesterolemia and the ACE1 genotype. Our work suggested that the ACE1-I/D might influence COVID-19 severity, but the effect was dependent on the hypertensive status. This result requires further validation in other large cohorts.

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.

Estabilidad del ARN viral en muestras clínicas para el diagnóstico viral / Stability of viral RNA in clinical specimens for viral diagnosis

No disponible

Merkel cell carcinoma caused by Merkel cell polyomavirus following trauma / Carcinoma de células de Merkel por poliomavirus de células de Merkel tras traumatismo

No disponible

Primary antimicrobial resistance rates and prevalence of Helicobacter pylori infection in the north of Spain. A 13-year retrospective study / Tasas de resistencia primaria a los antibióticos y prevalencia de la infección por Helicobacter pylori en el norte de España. Estudio retrospectivo de 13 años

Objective: Helicobacter pylori resistance to antimicrobial agents is on the rise and it is thus imperative to be aware of local resistance rates. The main objective of the present study was to describe the evolution of primary antimicrobial resistance in H. pylori, analysing its antibiotic susceptibility over a 13-year period in a region of northern Spain, as well as host-related factors. Patients and methods: Between 2004 and 2016 a total of 3426 patients who met the H. pylori eradication criteria underwent gastroscopy. The gastric biopsies were processed and those testing positive for H. pylori were identified and tested for clarithromycin, metronidazole and levofloxacin susceptibility using E-test. Results: H. pylori was isolated in 1604 (47%) patients, ranging from 63% (133/212) in 2004 to 39% (137/347) in 2016. Primary resistances to clarithromycin, metronidazole and levofloxacin were on average 19% (278/1116), 40% (572/865) and 17% (137/669), respectively. Clarithromycin resistance was 24% (167/686) in females and 15% (11/753) in males (p=0.0002); metronidazole resistance was 29% (72/246) in patients over 70 years compared to 42% (499/1190) in younger patients (p=0.0396); levofloxacin resistance increased with age, being 13% (57/439) in patients ≤55 years, 19% (46/236) for those between 56 and 70, and 26% (34/130) in patients >70 years (p=0.0087). Discussion: A decline in the prevalence of H. pylori infection was observed over the years, along with relatively high rates of primary resistance to clarithromycin, metronidazole and levofloxacin. Variations in resistance rates were found with sex and age

Objetivo: El aumento de la resistencia de Helicobacter pylori a los antibióticos hace indispensable conocer las tasas de resistencia locales. El principal objetivo de este estudio fue describir la evolución de la resistencia primaria de H. pylori a los antibióticos, analizando su sensibilidad durante un período de tiempo de 13 años en una región del norte de España, así como los factores asociados del huésped. Pacientes y métodos: Entre 2004 y 2016 se realizaron gastroscopias a 3.426 pacientes que cumplían criterios de erradicación de la infección por H. pylori. En las biopsias gástricas en las que se detectó crecimiento compatible con H. pylori se identificó este microorganismo y se testó la sensibilidad a claritromicina, metronidazol y levofloxacino mediante Etest(R). Resultados: Se aisló H. pylori en 1.604 (47%) pacientes, desde el 63% (133/212) en 2004 hasta el 39% (137/347) en 2016. Las resistencias primarias a claritromicina, metronidazol y levofloxacino fueron del 19% (278/1.116), 40% (572/865) y 17% (137/669), respectivamente. La resistencia a claritromicina fue mayor en mujeres: 24% (167/686) vs. 15% (11/753) (p=0,0002); la resistencia a metronidazol fue mayor en jóvenes: 29% (72/246) en >70 años vs. 42% (499/1.190) en ≤70 años (p=0,0396); la resistencia a levofloxacino aumentó con la edad de los pacientes: 13% (57/439) en <55 años, 19% (46/236) entre 56 y 70 años y 26% (34/130) en >70 años (p=0,0087). Discusión: Se observa una disminución en la prevalencia de la infección por H. pylori a lo largo de los años, con tasas relativamente altas de resistencia primaria a claritromicina, metronidazol y levofloxacino. Se encuentran variaciones en estas tasas de resistencia en función del sexo y de la edad de los pacientes

Infant Facial Paralysis Associated with Epstein-Barr Virus Infection.

BACKGROUND Peripheral facial paralysis is a clinical presentation which, in most cases, is benign. It is relatively frequent, although less so in pediatric patients, where clinical diagnosis is more difficult. This clinical condition can be congenital, neurological, infectious, neoplastic, traumatic, or metabolic in origin. CASE REPORT This report describes the case of a male infant of 23 months of age with peripheral facial paralysis due to Epstein-Barr virus (EBV) upper respiratory infection. A hemogram showed the presence of leukocytosis and lymphocytosis, and a peripheral blood smear indicated the presence of stimulated lymphocytes. Serological tests were compatible with recent EBV infection: IgM anti-VCA (capsid antigen) was positive, while IgG anti-VCA and anti-EBNA (nuclear antigen) were negative. EBV genome was detected in pharyngeal swab and in serum, where viral load was 5.08 log copies/1000 cells and 3.72 log copies/mL, respectively. CONCLUSIONS Whilst the most common cause of facial paralysis is idiopathic paralysis, such problems of the facial nerve may have many origins, including an infectious nature such as infection with viral agents. Rapid determination of the etiology of the problem allows the most appropriate management of the condition and quick follow-up to be implemented, which is essential for the evaluation of treatment response and the avoidance of permanent consequences.

Primary antimicrobial resistance rates and prevalence of Helicobacter pylori infection in the north of Spain. A 13-year retrospective study.

OBJECTIVE: Helicobacter pylori resistance to antimicrobial agents is on the rise and it is thus imperative to be aware of local resistance rates. The main objective of the present study was to describe the evolution of primary antimicrobial resistance in H. pylori, analysing its antibiotic susceptibility over a 13-year period in a region of northern Spain, as well as host-related factors. PATIENTS AND METHODS: Between 2004 and 2016 a total of 3426 patients who met the H. pylori eradication criteria underwent gastroscopy. The gastric biopsies were processed and those testing positive for H. pylori were identified and tested for clarithromycin, metronidazole and levofloxacin susceptibility using E-test. RESULTS: H. pylori was isolated in 1604 (47%) patients, ranging from 63% (133/212) in 2004 to 39% (137/347) in 2016. Primary resistances to clarithromycin, metronidazole and levofloxacin were on average 19% (278/1116), 40% (572/865) and 17% (137/669), respectively. Clarithromycin resistance was 24% (167/686) in females and 15% (11/753) in males (p=0.0002); metronidazole resistance was 29% (72/246) in patients over 70 years compared to 42% (499/1190) in younger patients (p=0.0396); levofloxacin resistance increased with age, being 13% (57/439) in patients ≤55 years, 19% (46/236) for those between 56 and 70, and 26% (34/130) in patients >70 years (p=0.0087). DISCUSSION: A decline in the prevalence of H. pylori infection was observed over the years, along with relatively high rates of primary resistance to clarithromycin, metronidazole and levofloxacin. Variations in resistance rates were found with sex and age.

Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, Yellow fever virus and West Nile virus.

Although certain mosquito-borne virus, such as Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Yellow fever virus (YFV) and West Nile virus (WNV), are an important public health concern in those countries where transmitter mosquitoes are endemic, several cases of travelers from those endemic countries have been recently reported in Europe. Thus, early diagnosis of these viruses is essential for patient management and adoption of preventive measures. An assay for the simultaneous detection of DENV, CHIKV, ZIKV, YFV and WNV based on a multiplex real-time (RT)-PCR and its usefulness for diagnosis in infection screenings and surveillance of arbovirus in non-endemic countries are described.

New clinical and seasonal evidence of infections by Human Parainfluenzavirus.

Human Parainfluenzaviruses (PIVs) account for a significant proportion of viral acute respiratory infections (ARIs) in children, and are also associated with morbidity and mortality in adults, including nosocomial infections. This work aims to describe PIV genotypes and their clinical and epidemiological distribution. Between December 2016 and December 2017, 6121 samples were collected, and submitted to viral culture and genomic quantification, specifically Parainfluenza 1-4 (PIV1-4), Influenza A and B, Respiratory Syncytial Virus (RSV) A and B, Adenovirus, Metapneumovirus, Coronavirus, Rhinovirus, and Enterovirus. Normalized viral load, as (log10) copies/103 cells, was calculated as virus Ct, determined by multiple qRT-PCR, as a function of the Ct of ß-globin. PIV was confirmed in 268 cases (4.37%), and linked to both upper and lower respiratory tract disease, being more frequent in children than in adults (5.23 and 2.43%, respectively). PIV1 and PIV3 were most common (31 and 32.5%, of total PIV positive samples, respectively), with distribution being similar in children and adults, as was viral load. PIV type was correlated with seasonality: PIV3 being more frequent in winter and spring, PIV1 in summer, and PIV 4 in fall. No correlation between vial load and clinical severity was found. Novel findings were that PIV viral load was higher in fall than in other seasons, and PIV4, classically linked to mild respiratory symptoms, was circulating, in children and adults, at all levels of symptoms throughout the year.

Human respiratory syncytial virus load normalized by cell quantification as predictor of acute respiratory tract infection.

Human respiratory syncytial virus (HRSV) is a common cause of respiratory infections. The main objective is to analyze the prediction ability of viral load of HRSV normalized by cell number in respiratory symptoms. A prospective, descriptive, and analytical study was performed. From 7307 respiratory samples processed between December 2014 to April 2016, 1019 HRSV-positive samples, were included in this study. Low respiratory tract infection was present in 729 patients (71.54%). Normalized HRSV load was calculated by quantification of HRSV genome and human ß-globin gene and expressed as log10 copies/1000 cells. HRSV mean loads were 4.09 ± 2.08 and 4.82 ± 2.09 log10 copies/1000 cells in the 549 pharyngeal and 470 nasopharyngeal samples, respectively (P < 0.001). The viral mean load was 4.81 ± 1.98 log10 copies/1000 cells for patients under the age of 4-year-old (P < 0.001). The viral mean loads were 4.51 ± 2.04 cells in patients with low respiratory tract infection and 4.22 ± 2.28 log10 copies/1000 cells with upper respiratory tract infection or febrile syndrome (P < 0.05). A possible cut off value to predict LRTI evolution was tentatively established. Normalization of viral load by cell number in the samples is essential to ensure an optimal virological molecular diagnosis avoiding that the quality of samples affects the results. A high viral load can be a useful marker to predict disease progression.