Direct Identification of Urinary Tract Pathogens from Urine Samples, Combining Urine Screening Methods and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy. Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000iflow cytometer, an automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triple-centrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microorganisms were misidentified, and noCandidaspp. were correctly identified. Regarding the data from autoanalyzers, the best bacteriuria cutoffs were 1,000 and 200 U/µl for UF-1000iand SediMax, respectively. It was concluded that the combination of a urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those containing Gram-negative bacteria.

Treatment of infections caused by carbapenemase-producing Enterobacterales

Antibiotic resistance is one of the main menaces to public and individual health worldwide. In the last two decades, an increase in the detection of arbapenem-resistant Enterobacterales has been reported. The treatment of infections caused by these strains is a therapeutic challenge. The use of carbapenems may be beneficial depending on MIC value and source of infection. New drugs, with different activity against the different classes of carbapenemases, are developed showing significant benefits. (AU)

Evaluation of the SediMax automated microscopy sediment analyzer and the Sysmex UF-1000i flow cytometer as screening tools to rule out negative urinary tract infections.

Urinary tract infections (UTI) are highly prevalent in nosocomial and community settings, and their diagnosis is costly and time-consuming. Screening methods represent an important advance towards the final UTI diagnosis, diminishing inappropriate treatment or clinical complications. Automated analyzers have been developed and commercialized to screen and rule out negative urine samples. The aim of this study was to evaluate two of these automated analyzers (SediMax, an automatic sediment analyzer and UF-1000i a flow cytometer) to predict negative urine cultures. A total of 1934 urine samples were analyzed. A very strong correlation for white blood cells (WBC) (rs: 0.928) and a strong correlation for bacteria (BAC) (rs: 0.693) were obtained. We also calculated optimal cut-off points for both autoanalyzers: 18 WBC/µL and 97 BAC/µL for SediMax (sensitivity=96.25%, specificity=63.04%, negative predictive value=97.97%), and 40 WBC/µL and 460 BAC/µL for UF-1000i (sensitivity=98.13%, specificity=79.16%, negative predictive value=99.18%). The use of SediMax and UF-1000i resulted in a 46.33% and 57.19% reduction of all samples cultured, respectively. In conclusion, both analyzers are good UTI screening tools in our setting.

Recurrent invasive pneumococcal disease in children: underlying clinical conditions, and immunological and microbiological characteristics.

PURPOSE: Clinical, immunological and microbiological characteristics of recurrent invasive pneumococcal disease (IPD) in children were evaluated, differentiating relapse from reinfection, in order to identify specific risk factors for both conditions. METHODS: All patients <18 years-old with recurrent IPD admitted to a tertiary-care pediatric center from January 2004 to December 2011 were evaluated. An episode of IPD was defined as the presence of clinical findings of infection together with isolation and/or pneumococcal DNA detection by Real-Time PCR in any sterile body fluid. Recurrent IPD was defined as 2 or more episodes in the same individual at least 1 month apart. Among recurrent IPD, we differentiated relapse (same pneumococcal isolate) from reinfection. RESULTS: 593 patients were diagnosed with IPD and 10 patients died. Among survivors, 23 episodes of recurrent IPD were identified in 10 patients (1.7%). Meningitis was the most frequent form of recurrent IPD (10 episodes/4 children) followed by recurrent empyema (8 episodes/4 children). Three patients with recurrent empyema caused by the same pneumococcal clone ST306 were considered relapses and showed high bacterial load in their first episode. In contrast, all other episodes of recurrent IPD were considered reinfections. Overall, the rate of relapse of IPD was 0.5% and the rate of reinfection 1.2%. Five out of 7 patients with reinfection had an underlying risk factor: cerebrospinal fluid leak (n = 3), chemotherapy treatment (n = 1) and a homozygous mutation in MyD88 gene (n = 1). No predisposing risk factors were found in the remainder. CONCLUSIONS: recurrent IPD in children is a rare condition associated with an identifiable risk factor in case of reinfection in almost 80% of cases. In contrast, recurrent IPD with pleuropneumonia is usually a relapse of infection.

Performance of a rapid multi-analyte 2-photon excitation assay in children with acute respiratory infection.

The purpose of this study is to evaluate the diagnostic performance of the novel 2-photon excitation-based mariPOC© Assay (ArcDia Laboratories, Turku, Finland) for antigen detection of respiratory viruses versus real-time polymerase chain reaction (PCR). The mariPOC Assay and 2 multiplex real-time PCR techniques were performed on nasopharyngeal samples from pediatric patients with suspicion of acute respiratory infection admitted to a children's hospital in Spain during October 2011 to January 2013. A total of 233 samples were studied. Sensitivities and specificities (95% confidence interval) of the mariPOC Assay were for respiratory syncytial virus (RSV), 78.4% (69.7-85.6) and 99.2% (96.3-100.0); influenza virus (IFV) A, 66.7% (26.2-94.0) and 99.6% (97.9-100.0); IFV-B, 63.6% (33.6-87.2) and 100.0% (98.7-100.0); human metapneumovirus (hMPV), 60.0% (34.5-81.9) and 100.0% (98.6-100.0); adenovirus (ADV), 12.5% (0.6-48.0) and 100.0% (98.7-100.0), respectively. The mariPOC Assay is a highly specific method for simultaneous detection of 8 respiratory viruses but has sensitivities that range from moderately high for RSV to moderate for IFV and hMPV and low for ADV.

Fungal biofilms: From bench to bedside / Biopelículas fúngicas: Del laboratorio a la práctica clínica

Biofilms cause recurrent invasive infections that are difficult to eradicate because of their high resistance to antimicrobials and host defence mechanisms. Fungal biofilm-related infections are associated with high mortality rates. Although current guidelines recommend catheter removal for catheter-related bloodstream infections due to Candida species, several studies have shown that the efficacy of the antifungal lock technique. The use of combinations of antifungal agents may improve the management of biofilm-related fungal infections and prevent the emergence of resistance associated with monotherapy. Since the presence of mixed bacterial-fungal biofilm infections is very prevalent, a combination of antibacterial and antifungal agents should be considered

Las infecciones relacionadas con biopelículas fúngicas se asocian con altas tasas de mortalidad. La infección asociada a catéteres son un ejemplo. Aunque las guías actuales recomiendan la retirada del catéter para tratar estas infecciones, varios estudios han demostrado la eficacia de la técnica de sellado antifúngico. El uso de combinaciones de agentes antifúngicos puede mejorar el pronóstico de las infecciones fúngicas relacionadas con biopelículas y prevenir la aparición de resistencias. Las infecciones asociadas a biopelículas mixtas (bacteria-hongo) son cada vez más frecuentes y requieren de un abordaje específico para conseguir su erradicación

Pneumococcal serotypes causing acute otitis media among children in Barcelona (1992-2011): emergence of the multiresistant clone ST320 of serotype 19A.

BACKGROUND: There is scarce information about changes in serotypes and clonal types of Streptococcus pneumoniae causing acute otitis media (AOM) in recent years, particularly in European countries. METHODS: Pneumococcal serotypes and clones from S. pneumoniae strains isolated from children with AOM who were attended at Hospital Sant Joan de Déu, Barcelona (1992 to 2011), were studied. Heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in June 2001. We defined 3 periods: prevaccine period 1992 to 2001, early vaccine period 2002 to 2006 and late vaccine period 2007 to 2011. RESULTS: There were 376 pneumococcal strains causing AOM, and 373 (99.2%) of them were serotyped. AOM caused by PCV7 serotypes declined significantly: 161 of 245 (65.7%) episodes in 1992 to 2001 versus 22 of 67 (32.8%) in 2002 to 2006 versus 8 of 61 (13.1%) in 2007 to 2011 P < 0.001. In the last period (2007 to 2011), the potential serotype coverage for the PCV10 was 16.4% and for the PCV13 was 68.9% (P < 0.001). Serotype 19A increased from 5.7% in 1992 to 2001 to 42.6% in 2007 to 2011 (P < 0.001). Among strains with penicillin minimal inhibitory concentration ≥0.12 µg/mL (n = 241), serotype 19A rose from 2.3% in the first period to 57.9 % in the last period (P < 0.001). The clonal-type ST320 was initially detected in 2005, and in the period 2007 to 2011, the ST320 was found in 72.7% of nonsusceptible serotype 19A isolates. CONCLUSIONS: Among children with AOM, a rapid expansion of the multiresistant clone ST320 expressing serotype 19A has been observed in Barcelona. The implementation of PCV13, which includes this serotype, may decrease the prevalence of AOM and reduce antimicrobial resistance.

Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries.

BACKGROUND: Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. METHODS: Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. RESULTS: Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). CONCLUSION: Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.

Antifungal activity against Candida biofilms.

Candida species have two distinct lifestyles: planktonic, and surface-attached communities called biofilms. Mature C. albicans biofilms show a complex three-dimensional architecture with extensive spatial heterogeneity, and consist of a dense network of yeast, hyphae, and pseudohyphae encased within a matrix of exopolymeric material. Several key processes are likely to play vital roles at the different stages of biofilm development, such as cell-substrate and cell-cell adherence, hyphal development, and quorum sensing. Biofilm formation is a survival strategy, since biofilm yeasts are more resistant to antifungals and environmental stress. Antifungal resistance is a multifactorial process that includes multidrug efflux pumps, target proteins of the ergosterol biosynthetic pathway. Most studies agree in presenting azoles as agents with poor activity against Candida spp. biofilms. However, recent studies have demonstrated that echinocandins and amphotericin B exhibit remarkable activity against C. albicans and Candida non-albicans biofilms. The association of Candida species with biofilm formation increases the therapeutic complexity of foreign body-related yeast infections. The traditional approach to the management of these infections has been to explant the affected device. There is a strong medical but also economical motivation for the development of novel anti-fungal biofilm strategies due to the constantly increasing resistance of Candida biofilms to conventional antifungals, and the high mortality caused by related infections. A better description of the extent and role of yeast in biofilms may be critical for developing novel therapeutic strategies in the clinical setting.