The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Skin manifestations during the COVID-19 pandemic in the pediatric emergency department.

BACKGROUND: The role of SARS-CoV-2 as the cause of chilblains in children remains a matter of debate but it is important to elucidate it for patient isolation and contact tracing. We sought to define the etiology, clinical presentation, time course, and outcomes of children presenting to the emergency department (ED) with cutaneous manifestations shortly after the first pandemic peak of COVID-19 in Spain. METHODS: A prospective, observational study in children <15 years of age evaluated for skin lesions in the EDs of three pediatric hospitals. Children underwent a comprehensive work-up including tests for SARS-CoV-2 antibodies and polymerase chain reaction (PCR), and serology and PCR tests for other viruses and bacteria. A 1 month follow-up visit was conducted. RESULTS: From April 14 through May 8, 2020, we enrolled 62 children. Of those, 34 had acro-ischemic skin lesions and 28 had a variety of skin rashes. Overall, 40% of children had mild systemic symptoms. Children with chilblains were older, had pain more frequently and a more prolonged duration of skin lesions, while those with non-specific rashes had fever more frequently. Lesions were resolved in 75% of children at follow up. Five patients demonstrated SARS-CoV-2 antibodies, and none tested positive with PCR. Three additional patients tested positive with PCR for rhinovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae. CONCLUSIONS: The number of ED visits for chilblains, which are rare in pediatrics, was high soon after the first peak of COVID-19 in Spain. The disease course was self-limited, outcomes were favorable, and the possibility of viral transmission was negligible as all patients tested negative for SARS-CoV-2 by PCR.

Evaluación de una prueba rápida para la detección de PBP2a en Staphylococcus aureus / Evaluation of a rapid assay for detection of PBP2a Staphylococcus aureus

INTRODUCCIÓN: En los últimos años se ha producido un incremento de las infecciones producidas por Staphylococcus aureus resistente a meticilina (SARM). En comparación con las producidas por S. aureus sensible a meticilina (SASM), las infecciones por SARM requieren estancias hospitalarias más prolongadas y presentan mayor mortalidad. La detección rápida de la resistencia a la meticilina por la adquisición del gen mecA que codifica la proteína fijadora de penicilina (PBP2a) es crucial para evitar la diseminación nosocomial e instaurar una correcta terapia antimicrobiana. Nos proponemos evaluar el test inmunocromatográfico rápido para la detección de PBP2a directamente de colonias de S. aureus, PBP2a SA Culture Colony Test(R) (ICPBP2a). MATERIAL Y MÉTODOS: En 107 cepas de S. aureus se estudió la resistencia a meticilina mediante las siguientes pruebas: el sistema automatizado Vitek2(R) (bioMérieux), CHROMagar MRSA II(R) (BD Becton Dickinson ), difusión con disco de cefoxitina, la ICPBP2a (AlereTM) y como método de referencia, la detección molecular del gen mecA. RESULTADOS: La sensibilidad y especificidad para las pruebas de detección fueron para la difusión en agar con disco de cefoxitina 100% y 100% respectivamente, Vitek2(R) 100% y 100%, CHROMagarTM MRSA II 100% y 96%, y la ICPBP2a 98,25% y 100%. CONCLUSIÓN: La inmunocromatografía para la detección de PBP2a es una técnica rápida, fácil y económica. Resulta muy útil para el manejo de brotes hospitalarios

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen causing both healthcare-associated and community-acquired infection. Rapid and accurate detection of this pathogen is crucial for the use of appropriate antimicrobial therapy and the control of nosocomial spread. METHODS: A total of 107 S. aureus strains were assayed for methicillin resistance: Vitek2(R) (bioMérieux), CHROMagarTM MRSA II (BD Becton Dickinson), disk diffusion in agar for cefoxitin 30 μg and immunochromatography PBP2a SA Culture Colony Test (AlereTM). The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. RESULTS: Sensitivity and specificity were: disk diffusion for cefoxitin 100% and 100% respectively, Vitek2(R) 100 and 100%, CHROMagarTM MRSA II 100 and 96%, and ICPBP2a detection 98,25% and 100%. CONCLUSION: ICPBP2a Culture Colony Test (AlereTM) is fast, efficient and economical technique for detection of penicillin binding protein 2a (PBP2a) from isolates. This assay is a useful tool for the management of hospital outbreaks

Evaluación de un ensayo inmunocromatográfico para la detección de carbapenemasa OXA-48 / Evaluation of an immunochromatographic test for the detection of OXA-48 carbapenemase

Introducción. La detección y diferenciación de los distintos tipos de carbapenemasas es crucial para el control y diseminación de las mismas. OXA-48 es la carbapenemasa más frecuente en España y en nuestro medio. El objetivo del estudio fue evaluar el nuevo test inmunocromatográfico OXA-48 Card letitest (Coris, BioConcept Belgium) para detectar esta carbapenemasa a partir de medios sólidos. Material y Métodos. Durante el último año se han aislado 151 cepas productoras de carbapenemasas, de las cuales 136 presentaban OXA-48 (126 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 5 Escherichia coli, 4 Enterobacter cloacae) y 15 productoras de otras carbapenemasas. Estas 15 cepas junto con otras 73 con distintos mecanismos de resistencia: 54 productoras de β-lactamasas de espectro extendido y 19 con otros mecanismos, fueron utilizadas como controles negativos. Resultados. Las 136 cepas portadoras de OXA-48 resultaron positivas en la prueba OXA-48 Card letitest y las 88 especies utilizadas como controles fueron negativos, por lo que la sensibilidad y especificidad de la prueba OXA-48 Card letitest fue del 100%. Discusión. La OXA-48 Card letitest resulta ser una prueba fácil, rápida, segura y barata (aproximadamente unos 6 Euros por test) que puede utilizarse en los laboratorios de Microbiología para confirmar la producción de carbapenemasa OXA-48 a partir de los aislamientos clínicos (AU)

Introduction. Detection and differentiation of various types of carbapenemases is crucial to their control and dissemination. OXA -48 is the most common carbapenemase in Spain and in our environment. The aim of this study is the evaluation of a new immunochromatographic test OXA-48 Card letitest (Coris, BioConcept Belgium) to detect this carbapenemase from solid media. Material and Methods. During the last year 151 strains of carbapenemase producing bacteria have been isolated, of which 136 were OXA-48 (126 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 5 Escherichia coli, 4 Enterobacter cloacae), and 15 producing other carbapenemases . These 15 strains with other 73 carrying other resistance mechanisms (54 extended-spectrum β-lactamases producers and 19 with other mechanisms) were used as negative controls. Results. One hundred and thirty six strains carrying OXA- 48 were positive with the test OXA-48 Card letitest and the 88 species used as controls were negative, resulting in a sensitivity and specificity of 100%. Discussion. The OXA-48 Card letitest is simple, quick, safe and cheap (approx. 6Euros/test) and can be used in microbiology laboratories to confirm the production of OXA-48 carbapenemase in clinical isolates (AU)

Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis.

BACKGROUND: Nucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods. METHODS: This single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene. RESULTS: The detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively. CONCLUSION: Real-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and complementary tool in the diagnosis of tuberculosis.

[Microbiological diagnosis of urinary tract infections]. / Diagnóstico microbiológico de las infecciones urinarias.

Urinary tract infections (UTI) are the most common infectious diseases observed in primary care; up to one-third of women will have at least one symptomatic UTI by age 24, and more than one-half of women will be affected by the end of life. In addition, UTIs represent 40% of nosocomial infections, and being usually associated with urinary catheters. Although urine cultures would not be indicated in all cases, these samples are the most abundant in the laboratories of clinical microbiology. Thus, the working protocols applied to these samples have an important impact in the performance of the laboratory. The samples are collected by mid stream urine, and 60-70% of them are negative culture. At present, several commercial systems have been introduced in order to simplify and automate this process. A urine culture with ≥ 10(5) CFU/ml has classically been considered as positive, although lower counts are valued in certain clinical settings. Factors related to this count e.g. methods to obtain urine, conservation of the sample or use of chemical preservatives as well as low counts are critical points to be discussed in detail. The development of antimicrobial resistance logically affects uropathogens, mainly Escherichia coli, which remains the most frequently isolated in urine cultures. The aim of this paper is to review the most innovating aspects influencing the microbiological diagnosis of UTI.

Diagnóstico microbiológico de las infecciones urinarias / Microbiological diagnosis of urinary tract infections

La infección del tracto urinario es la infección bacteriana más frecuente observada en el ámbito ambulatorio: 1 de cada 3 mujeres desarrollará una infección urinaria que requerirá tratamiento con antibióticos antes de los 24 años y, al menos, el 50% una infección del tracto urinario durante su vida. Además, las infecciones del tracto urinario representan el 40% de las infecciones nosocomiales, usualmente asociadas a sondajes urinarios. Aunque el cultivo de orina no estaría indicado en todos los casos, estas muestras son las más abundantes en los laboratorios de microbiología clínica, y la carga de trabajo que conllevan tiene una repercusión importante en el funcionamiento del laboratorio. Se trata fundamentalmente de orinas ambulatorias de “micción media”, de las cuales el 60-70% son cultivo negativo. En la actualidad existen sistemas comerciales disponibles con objeto de automatizar y simplificar su procesamiento. Clásicamente se han considerado positivas orinas con recuento ≥ 105 UFC/ml, aunque se valoran recuentos más bajos en determinadas situaciones clínicas. Factores relacionados con este recuento, como el modo de obtención de la orina, su conservación y el uso de conservantes químicos, así como el significado de estos bajos recuentos son puntos críticos que se analizarán detalladamente. El desarrollo de resistencias antimicrobianas afecta, lógicamente, a los uropatógenos, fundamentalmente Escherichia coli, que sigue siendo el más frecuentemente aislado. El objetivo de este texto es hacer una revisión de los aspectos más relevantes que influyen en el diagnóstico microbiológico de las infecciones del tracto urinario

Urinary tract infections (UTI) are the most common infectious diseases observed in primary care; up to one-third of women will have at least one symptomatic UTI by age 24, and more than one-half of women will be affected by the end of life. In addition, UTIs represent 40% of nosocomial infections, and being usually associated with urinary catheters. Although urine cultures would not be indicated in all cases, these samples are the most abundant in the laboratories of clinical microbiology. Thus, the working protocols applied to these samples have an important impact in the performance of the laboratory. The samples are collected by mid stream urine, and 60-70% of them are negative culture. At present, several commercial systems have been introduced in order to simplify and automate this process. A urine culture with ≥ 105 CFU/ml has classically been considered as positive, although lower counts are valued in certain clinical settings. Factors related to this count e.g. methods to obtain urine, conservation of the sample or use of chemical preservatives as well as low counts are critical points to be discussed in detail. The development of antimicrobial resistance logically affects uropathogens, mainly Escherichia coli, which remains the most frequently isolated in urine cultures. The aim of this paper is to review the most innovating aspects influencing the microbiological diagnosis of UTI

Use of MALDI-TOF MS for Identification of Nontuberculous Mycobacterium Species Isolated from Clinical Specimens.

The aim of this study was to compare the results obtained for identification by MALDI-TOF of nontuberculous mycobacteria (NTM) isolated in clinical samples with those obtained by GenoType Mycobacterium CM/AS (common mycobacteria/additional species). A total of 66 Mycobacterium isolates from various clinical specimens (mainly respiratory) were tested in this study. They were identified using MALDI-TOF Bruker from strains isolated in Lowenstein, following the recommended protocol of heat inactivation and extraction, and were simultaneously analyzed through hybridization by GenoType Mycobacterium from liquid culture MGIT. Our results showed that identification by MALDI-TOF was correct in 98.4% (65/66) of NTM isolated in our clinical practice (M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum, M. mucogenicum, M. kansasii, and M. scrofulaceum). MALDI-TOF was found to be an accurate, rapid, and cost-effective system for identification of mycobacteria species.

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

[Serum C-reactive protein as a marker of outcome and infection in critical care patients]. / Valor de la determinación de la proteína C reactiva como marcador pronóstico y de infección en pacientes críticos.

BACKGROUND AND OBJECTIVE: C-reactive protein (CRP) has been considered a marker for infection and an aid for diagnosing sepsis. We analyze the relation of CRP to infection and outcome in intensive care units (ICU) patients. PATIENTS AND METHOD: Prospective study on 77 ventilated patients. Expected short ICU stay or (suspected or confirmed) infection at admission were excluding criteria. 55 admissions after elective surgery were the controls. CRP measurement the first (CRP-1), third (CRP-3) and sixth (CRP-6) day of stay. APACHE II (Acute Physiology Score and Chronic Health Evaluation), SOFA (Sepsis-related Organ Failure Assessment), shock, respiratory or renal failure, leucocytes, platelets and albumin were registered. Follow-up until day 9 for infection and ICU discharge for outcome. RESULTS: CRP-1 in controls was 5.3 (3.9) mg/l and cases 67.8 (77.4) (p < 0.001). Shock on admission was related to CRP-1: patients in shock had higher CRP-1 levels (118.6 [82.8] vs 62.8 [75.6]; p = 0.06). 40.25% of cases developed infection, and CRP-1 levels were higher in this patients (88.8 [93.9] vs 53.8 [60.9]; p < 0.05). ROC area under curve was 0.6 with a sensibility of 23% and a specificity of 89% for a level of CRP-1 > 100. Mortality was 23.4% in cases and 1.8% in controls. Age, shock, APACHE II and SOFA were related to mortality, but CRP-1 did not. ROC area under curve for CRP-1 as mortality predictor in all patients was 0.62 (0.76 for APACHE II and 0.77 for SOFA) but only in cases was of 0.49 (0.69 for APACHE II and 0.67 for SOFA). CONCLUSIONS: CRP level on admission is an useful marker for early infection but not for outcome in critically ill patients admited to the ICU.

Valor de la determinación de la proteína C reactiva como marcador pronóstico y de infección en pacientes críticos / Serum C-reactive protein as a marker of outcome and infection in critical care patients

Fundamento y objetivo: Analizar la utilidad de la proteína C reactiva (PCR) como marcador pronóstico y de infección en pacientes ingresados en unidades de cuidados intensivos (UCI). Pacientes y método: Se ha realizado un estudio prospectivo en 77 pacientes ventilados mecánicamente sin infección (sospechada o confirmada) en el momento del ingreso; el grupo control estuvo formado por 55 ingresos tras cirugía electiva. Determinamos el valor de PCR los días 1 (PCR-1), 4 (PCR-4) y 7 (PCR-7). Se registraron el APACHE-II (Acute Physiology Score and Chronic Health Evaluation) y SOFA (Sepsis-related Orgam Failure Assessment) al ingreso y la presencia de shock, fallo respiratorio o renal, así como la cifra de leucocitos, plaquetas y albúmina sérica durante el seguimiento (10 días para el análisis de infecciones y hasta el alta de la UCI para el del pronóstico). Resultados: El valor medio (desviación estándar) de la PCR-1 en los controles fue de 5,3 (3,9) mg/l, frente a 67,8 (77,4) mg/l en los casos (p < 0,001). Los casos con shock en el momento del ingreso presentaron valores más elevados de PCR-1 (118,6 [82,8] frente a 62,8 [75,6] mg/l, p = 0,06). El 40,25% de los casos desarrolló infección y presentó valores de PCR-1 más elevados (88,8 [93,9] comparado con 53,8 [60,9] mg/l, p < 0,05). La sensibilidad fue del 23% y la especificidad del 89% para un valor de PCR-1 superior a 100 (area bajo la curva de 0,6). Las mortalidad en los casos fue del 23,4%. La PCR-1 no se relacionó con el pronóstico en este grupo: el área bajo la curva para PCR-1 mayor de 100 como predictor de mortalidad en toda la población fue de 0,62, pero en los casos fue sólo de 0,49 (0,69 para APACHE-II y 0,67 para SOFA). Conclusiones: El valor sérico de la PCR en el momento del ingreso es un marcador temprano de infección pero no es útil como marcador pronóstico en pacientes críticos sometidos a ventilación mecánica al ingresar en la UCI