Dynamics of respiratory viruses other than SARS-CoV-2 during the COVID-19 pandemic in Madrid, Spain.

The COVID-19 pandemic and the implemented control measures have impacted the circulation of respiratory-transmitted pathogens. In this report, we present data from a retrospective study that included 17,883 specimens conducted between 2018 and 2022 in our facility, describing the dynamics of circulation of the main respiratory viruses. We observed a significant decrease in all viral detections (other than SARS-CoV-2) starting from March 2020. However, rhinovirus maintained comparable levels to the pre-pandemic period. Additionally, influenza viruses were not detected during the 2020-2021 season, and respiratory syncytial virus (RSV) exhibited a shift in its seasonality, with an epidemic peak occurring in the summer of 2021.

Rapid screening of SARS-CoV-2 variants, a key tool for pandemic surveillance.

The utility of reverse transcription-polymerase chain reaction (RT-PCR) in analysis SARS-COV-2 variants was evaluated. RT-PCR tests were used to analyse the majority of new SARS-CoV-2 cases (n = 9315) in a tertiary hospital (Madrid, Spain) throughout 2021. Subsequently, whole genome sequencing (WGS) was conducted on 10.8% of these samples (n = 1002). Notably, the Delta and Omicron variants emerged rapidly. There were no discrepancies between RT-PCR and WGS results. Continuous surveillance of SARS-CoV-2 variants is essential, and RT-PCR is a highly useful method, specially during periods of high COVID-19 incidence. This feasible technique can be implemented in all SARS-CoV-2 laboratories. However, WGS remains the gold standard method for comprehensive detection of all existing SARS-CoV-2 variants.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant / Primera confirmación de importación y transmisión en España de la variante B.1.1.7 del SARS-CoV-2 recientemente identificada

Introduction: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and methods: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. Results: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. Conclusion: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.(AU)

Introducción: Recientemente, ha surgido en Reino Unido una nueva variante de SARS-CoV-2, VOC202012/01, que origina el linaje B.1.1.7. Su rápida distribución en Reino Unido ha alertado a otros países a vigilar su presencia. Material y métodos: El rastreo inicial de la variante B.1.1.7 se basó en la ausencia de amplificación del gen S en el ensayo TaqPath, causado por la deleción 69/70. Todos los casos candidatos de corresponder a la variante B.1.1.7 con este criterio fueron posteriormente confirmados por secuenciación de genoma completo. Resultados: Describimos los primeros 3 casos importados de esta variante, desde Londres hasta Madrid, con la posterior transmisión domiciliaria de uno de estos casos a 3 familiares y, adicionalmente, los 2 primeros casos con la variante sin vínculo epidemiológico con Reino Unido. Uno de los casos requirió hospitalización. En todos los casos el criterio de no amplificación del gen S identificó con precisión la variante B.1.1.7, como demostró posteriormente la presencia de las 17 mutaciones marcadoras de este linaje. Conclusión: Las primeras identificaciones de la variante B.1.1.7 de SARS-CoV-2 indican un papel solapante de las introducciones independientes desde Reino Unido, con eventos de transmisión comunitaria, incluso desde los primeros momentos de la presencia de esta variante en nuestro país.(AU)

Strain-to-strain variability among Staphylococcus aureus causing prosthetic joint infection drives heterogeneity in response to levofloxacin and rifampicin.

INTRODUCTION: Levofloxacin and rifampicin are the preferred treatment for prosthetic joint infection (PJI) caused by Staphylococcus aureus, especially when managed with implant retention (DAIR). However, a significant variability of success has been reported, which could be related to intrinsic characteristics of the microorganism. Our aim was to evaluate the variability in the anti-biofilm response to levofloxacin and rifampicin in a clinical collection of S. aureus. MATERIAL AND METHODS: Eleven levofloxacin- and rifampicin-susceptible S. aureus isolates causing PJI managed with DAIR were included. Levofloxacin, rifampicin and levofloxacin + rifampicin were tested in an in vitro static biofilm model in microtitre plates, where 48 h biofilms were challenged with antimicrobials during 24 h. Additionally, two genetically similar strains were tested in the CDC Biofilm Reactor, where 48 h biofilms were treated during 56 h. Antimicrobial activity was assessed by viable biofilm-embedded cells recount, and by crystal violet staining. RESULTS: All antimicrobial regimens showed significant anti-biofilm activity, but a notable scattering in the response was observed across all strains (inter-strain coefficient of variation for levofloxacin, rifampicin and levofloxacin + rifampicin of 22.8%, 35.8% and 34.5%, respectively). This variability was tempered with the combination regimen when tested in the biofilm reactor. No correlation was observed between the minimal biofilm eradicative concentration and the antimicrobial activity. Recurrent S. aureus isolates exhibited higher biofilm-forming ability compared with strains from resolved infections (7.6 log10 cfu/cm2±0.50 versus 9.0 log10 cfu±0.07). CONCLUSIONS: Significant variability may be expected in response to levofloxacin and rifampicin among biofilm-embedded S. aureus. A response in the lower range, together with other factors of bad prognosis, could be responsible of treatment failure.

Microbiological and Molecular Features Associated with Persistent and Relapsing Staphylococcus aureus Prosthetic Joint Infection.

BACKGROUND: Persistent and relapsing prosthetic joint infection (PJI) due to Staphylococcus aureus presents a clinical challenge. This study aimed to provide an extensive description of phenotypic and genomic changes that could be related to persistence or relapse. METHODS: Initial and second S. aureus isolates from 6 cases of persistent and relapsing PJI, along with clinical isolates from 8 cases, with favorable outcome were included. All isolates were studied by phenotypic and genotypic approaches. RESULTS: Recurrent S. aureus isolates exhibited a significant increase in adhesive capacity, invasion and persistence compared to resolved isolates. No association was found for the presence or absence of certain genes with the persistence or relapse of PJI. All sequential isolates showed identical sequence type (ST). Resistance gene loss during the infection and a great diversity of variants in different virulence genes between the pair of strains, mainly in genes encoding adhesins such as fnbA, were observed. CONCLUSIONS: S. aureus-caused relapse and persistence PJI is associated with bacterial phenotypical and genotypical adaptation. The main paths of adaptation were persistence in the intracellular compartment, and the loss of antibiotic resistance genes and variant acquisition, especially in genes encoding adhesins.

Clinical presentation and virological assessment of confirmed human monkeypox virus cases in Spain: a prospective observational cohort study.

BACKGROUND: In May, 2022, several European countries reported autochthonous cases of monkeypox, which rapidly spread globally. Early reports suggest atypical presentations. We aimed to investigate clinical and virological characteristics of cases of human monkeypox in Spain. METHODS: This multicentre, prospective, observational cohort study was done in three sexual health clinics in Madrid and Barcelona, Spain. We enrolled all consecutive patients with laboratory-confirmed monkeypox from May 11 to June 29, 2022. Participants were offered lesion, anal, and oropharynx swabs for PCR testing. Participant data were collected by means of interviews conducted by dermatologists or specialists in sexually transmitted infections and were recorded using a standard case report form. Outcomes assessed in all participants with a confirmed diagnosis were demographics, smallpox vaccination, HIV status, exposure to someone with monkeypox, travel, mass gathering attendance, risk factors for sexually transmitted infections, sexual behaviour, signs and symptoms on first presentation, virological results at multiple body sites, co-infection with other sexually transmitted pathogens, and clinical outcomes 14 days after the initial presentation. Clinical outcomes were followed up until July 13, 2022. FINDINGS: 181 patients had a confirmed monkeypox diagnosis and were enrolled in the study. 166 (92%) identified as gay men, bisexual men, or other men who have sex with men (MSM) and 15 (8%) identified as heterosexual men or heterosexual women. Median age was 37·0 years (IQR 31·0-42·0). 32 (18%) patients reported previous smallpox vaccination, 72 (40%) were HIV-positive, eight (11%) had a CD4 cell count less than 500 cells per µL, and 31 (17%) were diagnosed with a concurrent sexually transmitted infection. Median incubation was 7·0 days (IQR 5·0-10·0). All participants presented with skin lesions; 141 (78%) participants had lesions in the anogenital region, and 78 (43%) in the oral and perioral region. 70 (39%) participants had complications requiring treatment: 45 (25%) had a proctitis, 19 (10%) had tonsillitis, 15 (8%) had penile oedema, six (3%) an abscess, and eight (4%) had an exanthem. Three (2%) patients required hospital admission. 178 (99%) of 180 swabs from skin lesions collected tested positive, as did 82 (70%) of 117 throat swabs. Viral load was higher in lesion swabs than in pharyngeal specimens (mean cycle threshold value 23 [SD 4] vs 32 [6], absolute difference 9 [95% CI 8-10]; p<0·0001). 108 (65%) of 166 MSM reported anal-receptive sex. MSM who engaged in anal-receptive sex presented with proctitis (41 [38%] of 108 vs four [7%] of 58, absolute difference 31% [95% CI 19-44]; p<0·0001) and systemic symptoms before the rash (67 [62%] vs 16 [28%], absolute difference 34% [28-62]; p<0·0001) more frequently than MSM who did not engage in anal-receptive sex. 18 (95%) of 19 participants with tonsillitis reported practising oral-receptive sex. The median time from onset of lesions to formation of a dry crust was 10 days (IQR 7-13). INTERPRETATION: In our cohort, monkeypox caused genital, perianal, and oral lesions and complications including proctitis and tonsillitis. Because of the variability of presentations, clinicians should have a low threshold for suspicion of monkeypox. Lesion swabs showed the highest viral loads, which, combined with the history of sexual exposure and the distribution of lesions, suggests close contact is probably the dominant transmission route in the current outbreak. FUNDING: None.

Detection of BK polyomavirus genotypes to predict the development of BK polyomavirus-associated complications in kidney transplant recipients: A retrospective analysis.

OBJECTIVES: This study focused on the role that BK polyomavirus (BKPyV) genotypes can play in the development of BKPyV-associated complications in renal transplant recipients. METHODS: A retrospective observational study (January 2015 to April 2018) was conducted by analyzing BKPyV genotypes in 180 blood samples with detectable BKPyV viral load (VL) > 1000 copies/mL, from 63 renal transplant recipients. VL and BKPyV genotypes detections were based on real-time PCR (rt-PCR)-specific assays. RESULTS: Forty-four patients (44/63 [69.8%]) were men, and the median age was 55.0 (interquartile range 49.0-66.0 years). Eleven patients had clinical manifestations (11/63 [17.5%]). The most frequently detected genotypes were I (14/63 [22.2%]) and II (13/63 [20.6%]). Half of the patients (33/63 [52.4%]) had a mixed genotype, most with genotypes I and II (25/33 [75.8%]). Patients with infection by mixed genotypes showed VLs that were detected earlier (in the first year after transplantation) than those with a single genotype (25/33 [75.8%] vs 13/30 [43.3%], P = .009) and demonstrated greater risk of developing clinical manifestations associated with BKPyV (odds ratio 12.609, 95% confidence interval 1.503-105.807). Moreover, patients with first BKPyV VL > 10 000 copies/mL more frequently presented mixed genotypes (12/16 [75.0%] vs 21/47 [44.7%], P = .036). CONCLUSIONS: The probability of developing clinical manifestations is higher in infections by mixed genotypes. Therefore, the detection of BKPyV genotypes by rt-PCR can provide relevant information to stratify patients' risk of BKPyV-associated complications and guide the clinical management of BKPyV infection in kidney transplant recipients.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Multicenter evaluation of the Panbio™ COVID-19 rapid antigen-detection test for the diagnosis of SARS-CoV-2 infection.

OBJECTIVES: The standard RT-PCR assay for coronavirus disease 2019 (COVID-19) is laborious and time-consuming, limiting testing availability. Rapid antigen-detection tests are faster and less expensive; however, the reliability of these tests must be validated before they can be used widely. The objective of this study was to determine the performance of the Panbio™ COVID-19 Ag Rapid Test Device (PanbioRT) (Abbott) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swab specimens. METHODS: This prospective multicentre study was carried out in ten Spanish university hospitals and included individuals with clinical symptoms or epidemiological criteria of COVID-19. Only individuals with ≤7 days from the onset of symptoms or from exposure to a confirmed case of COVID-19 were included. Two nasopharyngeal samples were taken to perform the PanbioRT as a point-of-care test and a diagnostic RT-PCR test. RESULTS: Among the 958 patients studied, 325 (90.5%) had true-positive results. The overall sensitivity and specificity for the PanbioRT were 90.5% (95%CI 87.5-93.6) and 98.8% (95%CI 98-99.7), respectively. Sensitivity in participants who had a threshold cycle (CT) < 25 for the RT-PCR test was 99.5% (95%CI 98.4-100), and in participants with ≤5 days of the clinical course it was 91.8% (95%CI 88.8-94.8). Agreement between techniques was 95.7% (κ score 0.90; 95%CI 0.88-0.93). CONCLUSIONS: The PanbioRT performs well clinically, with even more reliable results for patients with a shorter clinical course of the disease or a higher viral load. The results must be interpreted based on the local epidemiological context.

Genotypic and Phenotypic Characteristics of Staphylococcus aureus Prosthetic Joint Infections: Insight on the Pathogenesis and Prognosis of a Multicenter Prospective Cohort.

BACKGROUND: Staphylococcus aureus is the leading cause of prosthetic joint infection (PJI). Beyond the antibiogram, little attention has been paid to the influence of deep microbiological characteristics on patient prognosis. Our aim was to investigate whether microbiological genotypic and phenotypic features have a significant influence on infection pathogenesis and patient outcome. METHODS: A prospective multicenter study was performed, including all S. aureus PJIs (2016-2017). Clinical data and phenotypic (agr functionality, ß-hemolysis, biofilm formation) and genotypic characteristics of the strains were collected. Biofilm susceptibility to antimicrobials was investigated (minimal biofilm eradication concentration [MBEC] assay). RESULTS: Eighty-eight patients (39.8% men, age 74.7 ±â€…14.1 years) were included. Forty-five had early postoperative infections (EPIs), 21 had chronic infections (CPIs), and 19 had hematogenous infections (HIs). Twenty (22.7%) were caused by methicillin-resistant S. aureus. High genotypic diversity was observed, including 16 clonal complexes (CCs), with CC5 being the most frequent (30.7%). agr activity was greater in EPI than CPI (55.6% vs 28.6%; P = .041). Strains causing EPI were phenotypically and genotypically similar, regardless of symptom duration. Treatment failure (36.5%) occurred less frequently among cases treated with implant removal. In cases treated with debridement and implant retention, there were fewer failures among those who received combination therapy with rifampin. No genotypic or phenotypic characteristics predicted failure, except vancomycin minimal inhibitory concentration ≥1.5 mg/L (23.1% failure vs 3.4%; P = .044). MBEC50 was >128 mg/L for all antibiotics tested and showed no association with prognosis. CONCLUSIONS: S. aureus with different genotypic backgrounds is capable of causing PJI, showing slight differences in clinical presentation and pathogenesis. No major microbiological characteristics were observed to influence the outcome, including MBEC.

BK virus viral load: analysis of the requests received by the microbiology laboratory and clinical involvement of the issued results.

Automation of viral diagnosis has led to an increase of BK virus (BKV) viral load (VL) requests. The aim of this study was to assess the suitability of serum creatinine (SCr) for controlling the demand and to study the clinical characteristics of BKV infection. This is a retrospective study including patients with BKV VL request during April-July 2017. Clinical records and SCr were analyzed. Five hundred samples from 333 patients were included; 61.4% of samples were from males (55.5 ± 14.8 years), and all belonged to transplant recipients (86.4% renal). BKV VL was detectable in 40 samples (8.0%) from 23 patients (6.9%), who presented high SCr (100% vs. 90.9%, P = 0.038). Most of detectable VLs (62.5%) belonged to patients in their first year post-transplant. Six patients with detectable VL (26.1%) developed clinical manifestations, most of them (83.3%) had a first BKV VL greater than 10,000 copies/mL (P = 0.001). In conclusion, SCr would be useful to identify suitable specimens for BKV VL testing without missing cases.

Intraosteoblastic activity of levofloxacin and rifampin alone and in combination against clinical isolates of meticillin-susceptible Staphylococcus aureus causing prosthetic joint infection.

BACKGROUND: Staphylococcus aureus may invade and persist intracellularly in prosthetic joint infections (PJIs). Despite optimized treatments with levofloxacin plus rifampin, the intracellular reservoir may lead to infection relapse. This study assessed the intracellular activity of levofloxacin and rifampin in an in-vitro model of human osteoblastic infection. METHODS: Ten meticillin-susceptible S. aureus strains were used to infect osteoblastic MG63 cells. Osteoblasts were challenged with rifampin and levofloxacin at cortical and cancellous bone concentrations. Efficacy was measured as the intracellular counts of colony-forming units (log10CFU) compared with untreated controls. The emergence of small colony variants (SCVs) was determined, and the results were stratified according to the patient's prognosis (six cured and four with persistence/relapse). RESULTS: All regimes led to a significant decrease in CFU count compared with controls (1-2 log10CFU). Levofloxacin was the most effective treatment at both cortical and cancellous bone concentrations (-2.4 to -1.9 log10CFU, respectively). The addition of rifampin to levofloxacin did not improve performance (-1.9 log10CFU for cortical concentration and -1.8 log10 CFU for cancellous concentration). An increase in SCVs was observed in the presence of rifampin. The efficacy of antimicrobials was higher and the formation of SCVs was lower against strains belonging to PJIs with a favourable outcome. CONCLUSIONS: Levofloxacin plus rifampin had good intracellular activity against S. aureus. However, from the intracellular perspective, the addition of rifampin to levofloxacin showed no benefit but could account for an increased number of SCVs.

Influence of viral load in the outcome of hospitalized patients with influenza virus infection.

The role of viral load in the outcome of patients requiring hospital admission due to influenza is not well established. We aim to assess if there is an association between the viral load and the outcome in hospitalized patients with a confirmed influenza virus infection. A retrospective observational study including all adult patients who were hospitalized in our center with a confirmed influenza virus infection from January to May 2016. Viral load was measured by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) cycle threshold (Ct) value on upper respiratory tract samples. Its value was categorized into three groups (low Ct, ≤ 20; intermediate Ct, > 20-30; and high Ct, > 30). Two hundred thirty-nine patients were included. Influenza A/H1N1pdm09 was isolated in 207 cases (86.6%). The mean Ct value was 26.69 ± 5.81. The viral load was higher in the unvaccinated group when compared with the vaccinated patients (Ct 25.17 ± 5.55 vs. 27.58 ± 4.97, p = 0.004). Only 27 patients (11.29%) presented a high viral load. Patients with a high viral load more often showed abnormal findings on chest X-ray (p = 0.015) and lymphopenia (p = 0.097). By contrast, there were no differences between the three groups (according to viral load), in associated pneumonia, respiratory failure, need for mechanical ventilation, sepsis, or in-hospital mortality. Our findings suggest that in patients admitted to the hospital with confirmed influenza virus infection (mostly A/H1N1pdm09), a high viral load is associated with a higher presence of abnormal findings on chest X-ray but not with a significant worse prognosis. In these cases, standardized quantitative PCR could be useful.

Daptomycin Plus Fosfomycin as Salvage Therapy in a Difficult-to-Treat Total Femoral Replacement Infection.

The highly active anti-biofilm combination of daptomycin plus fosfomycin was successfully used in a difficult-to-treat infection of a total femoral replacement caused by multi-drug resistant Staphylococcus epidermidis in a 79-year-old woman. There was no need to remove the orthopedic hardware, and the patient is currently pain free and able to walk.

Molecular Epidemiology of Staphylococcus aureus Bacteremia: Association of Molecular Factors With the Source of Infection.

Staphylococcus aureus bacteremia (SAB) is associated with high morbidity and mortality, which varies depending on the source of infection. Nevertheless, the global molecular epidemiology of SAB and its possible association with specific virulence factors remains unclear. Using DNA microarrays, a total of 833 S. aureus strains (785 SAB and 48 colonizing strains) collected in Spain over a period of 15 years (2002-2017) were characterized to determine clonal complex (CC), agr type and repertoire of resistance and virulence genes in order to provide an epidemiological overview of CCs causing bloodstream infection, and to analyze possible associations between virulence genes and the most common sources of bacteremia. The results were also analyzed by acquisition (healthcare-associated [HA] and community-acquired [CA]), methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains, and patient age (adults vs. children). Our results revealed high clonal diversity among SAB strains with up to 28 different CCs. The most prevalent CCs were CC5 (30.8%), CC30 (20.3%), CC45 (8.3%), CC8 (8.4%), CC15 (7.5%), and CC22 (5.9%), which together accounted for 80% of all cases. A higher proportion of CC5 was found among HA strains than CA strains (35.6 vs. 20.2%, p < 0.001). CC5 was associated with methicillin resistance (14.7 vs. 79.4%, p < 0.001), whereas CC30, CC45, and CC15 were correlated with MSSA strains (p < 0.001). Pathogen-related molecular markers significantly associated with a specific source of bacteremia included the presence of sea, undisrupted hlb and isaB genes with catheter-related bacteremia; sed, splE, and fib genes with endocarditis; undisrupted hlb with skin and soft tissue infections; and finally, CC5, msrA resistance gene and hla gene with osteoarticular source. Our study suggests an association between S. aureus genotype and place of acquisition, methicillin resistance and sources of bloodstream infection, and provides a valuable starting point for further research insights into intrinsic pathogenic mechanisms involved in the development of SAB.

Sub-inhibitory concentrations of oxacillin modify the expression of agr locus in Staphylococcus aureus clinical strains belonging to different clonal complexes.

BACKGROUND: The ability of Staphylococcus aureus to invade tissues and cause an infectious disease is the result of a multi-factorial process supported by the huge number of virulence factors inherent to this microorganism tightly regulated by the accessory gene regulator (agr). During antimicrobial therapy bacteria may be exposed to sub-inhibitory concentrations (subMICs) of antibiotics that may trigger transcriptional changes that may have an impact on the pathogenesis of infection. The objective of this study was to investigate the effect of oxacillin sub-MICs on agr system expression as the key component in the regulation of virulence in methicillin-susceptible (MSSA) and -resistant S. aureus (MRSA) strains. Furthermore, we studied the genetic basis of the agr locus and their potential association with the expression levels. METHODS: We have examined the expression of RNAIII and agrA mRNA as biomarkers for agr expression in the presence and absence of oxacillin subMICs in 10 MSSA and 4 MRSA clinical strains belonging to 5 clonal complexes (CC45-agrI, CC8-agrI, CC5-agrII, CC15-agrII and CC30-agrIII) causing endovascular complications. The DNA sequences of agr locus were obtained by whole genome sequencing. RESULTS: Our results revealed that exposure to subMICs of oxacillin had an impact on agr locus expression modifying the relative levels of expression with increases in 11 strains and with decreases in 3 strains. Thereby, the exposure to subMICs of oxacillin resulted in higher levels of expression of agr in CC15 and CC45 and lower levels in CC30. We also observed the presence of mutations in agrC and agrA in 13/14 strains with similar mutation profiles among strains within individual CCs except for strains of CC5. Although, agr expression levels differed among strains within CCs, the presence of these mutations was associated with differences in agr expression levels in most cases. CONCLUSIONS: Changes in agr expression induced by exposure to oxacillin subMICs should be considered because they could lead to changes in the virulence modulation and have an adverse effect on the course of infection, especially in certain clonal complexes.

The value of xpert MTB/RIF-generated CT values for predicting the smear status of patients with pulmonary tuberculosis.

BACKGROUND: Smear microscopy is used to assess the patient's infectiousness at the time of initial diagnosis of pulmonary tuberculosis. However, its limited sensitivity and specificity highlights the need for new diagnostic strategies. The aim of our study was to assess the diagnostic accuracy of GX Ct value as a predictor of smear status and its usefulness to quantify mycobacterial load. METHODS: All GX-positive sputum samples during a seven-year period were included in the study. Correlations among Ct values, smear status and TTD on liquid culture were calculated. An optimal Ct value for ruling in infectious patients was established. Clinical and radiological variables were also analyzed. RESULTS: Sixty-eight samples from 65 patients were included. Ct value and TTD yielded a positive correlation (ρ = 0.714; p < 0.05), while Ct and smear grade yielded an inverse correlation (r = -0.71). An optimal Ct value for ruling in smear positive patients was established at 21.1 cycles (90.5% sensitivity, 61% specificity, 81% PPV and 78% NPV). CONCLUSIONS: Our study confirms the value of GX Ct levels for quantifying mycobacterial load and demonstrates the added value of Ct as a predictor of positive smear status, especially at Ct values below 21.

Pathogenic Characteristics of Staphylococcus aureus Endovascular Infection Isolates from Different Clonal Complexes.

Staphylococcus aureus is a major cause of bacteremia and, even with appropriate clinical management, causes high morbidity, and mortality due to its involvement in endovascular complications and metastatic infections. Through different pathogenic in vivo and in vitro models we investigated the behavior of S. aureus most relevant clonal complexes (CCs) causing endovascular complications. We analyzed 14 S. aureus strains representing CC5, CC8, CC15, CC30, and CC45 that caused endovascular complications, including methicillin susceptible and resistant isolates and strains with different functionality of the agr global regulator. Their adherence to collagen, interaction with the endothelium, resistance to immune attack, capacity to form biofilm and virulence in the Galleria mellonella model were analyzed. CC30 and CC45 showed greater adhesion to collagen and CC8 showed a trend towards higher rate of intracellular persistence in endothelial cells. All CCs exhibited similar tolerance to neutrophil antimicrobial peptide hNP-1 and were capable of forming biofilms under static conditions. The virulence assay in the G. mellonella model demonstrated that CC15 and CC30 were the most and least virulent, respectively. The analysis of the genomic sequences of the most relevant virulence genes identified some CC15 specific gene patterns (absence of enterotoxins and sak gene) and variants (mainly in leucocidins and proteases), but did not reveal any gene or variant that could be responsible for the increased virulence detected for CC15 strains. Even though all the CCs were capable of causing endovascular complications, our results showed that different CCs are likely to produce these complications through different mechanisms which, if confirmed in more sophisticated models, would indicate the need to more specific management and therapeutic approaches.