Comparing Two Automated Techniques for the Primary Screening-Out of Urine Culture.

Urinary tract infection is the most common human infection with a high morbidity. In primary care and hospital services, conventional urine culture is a key part of infection diagnosis but results take at least 24 h. Therefore, a rapid and reliable screening method is still needed to discard negative samples as quickly as possible and to reduce the laboratory workload. In this aspect, this study aims to compare the diagnostic performance between Sysmex UF-1000i and FUS200 systems in comparison to urine culture as the gold standard. From March to June 2016, 1,220 urine samples collected at the clinical microbiology laboratory of the "Miguel Servet" hospital were studied in parallel with both analysers, and some technical features were evaluated to select the ideal equipment. The most balanced cut-off values taking into account bacteria or leukocyte counts were 138 bacteria/µL or 119.8 leukocyte/µL for the UF-1000i (95.3% SE and 70.4% SP), and 5.7 bacteria/µL or 4.3 leukocyte/µL for the FUS200 (95.8% SE and 44.4% SP). The reduction of cultured plates was 37.4% with the FUS200 and 58.3% with the UF-1000i. This study shows that both techniques improve the workflow in the laboratory, but the UF-1000i has the highest specificity at any sensitivity and the FUS200 needs a shorter processing time.

Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis.

Antimicrobial photodynamic therapy (aPDT) has shown to exert a bactericidal effect against Streptococcus sanguinis and Streptococcus mutans. However, this efficacy has been reported for either type of bacteria separately. Bacterial suspensions of both strains, separately or together, were treated with concentrations of methylene blue (MB) and rose bengal (RB). Suspensions were irradiated with a light-emitting diode lamp (λ center at 625nm for MB and λ center at 515nm for RB) using a fluence of 18J/cm2. RB-aPDT at concentrations of 0.16-0.62 and 0.16-0.31µg/mL, and MB-aPDT at concentrations of 0.62-1.25 and 0.31-1.25µg/mL inhibited the growth of S. mutans and S. sanguinis respectively by 6 log10. In suspensions of both strains together, the same 6 log10 reduction in bacterial growth was achieved using the same concentrations of each photosensiziser. In conclusion, RB-aPDT and MB-aPDT appear to exert the same bactericidal effect against suspensions of S. sanguinis and S. mutans either for single strain treatment or for samples constituted by both bacteria mixed together. RB shows to be slightly more efficient than MB.

Towards the early detection of ß-lactamase-producing Enterobacteriaceae by MALDI-TOF MS analysis.

Objectives: Development of a novel MALDI-TOF MS-based method for the rapid detection of ESBL-producing Enterobacteriaceae. Methods: The method was evaluated in terms of sensitivity, specificity and turnaround time regarding the antibiotic used (cefotaxime, ceftazidime, ceftriaxone, cefpodoxime or cefepime) and the performance of the automated MBT STAR-BL software (Bruker Daltonik GmbH, Germany) relative to qualitative interpretation of spectra for detecting ß-lactamase resistance by MALDI-TOF MS (Bruker Daltonik) in a collection of 11 isogenic Escherichia coli control strains expressing different ß-lactamases. Finally, for clinical validation, ß-lactamase activity was determined under previously evaluated conditions in 100 clinical isolates previously characterized by PCR and sequencing. Results: Clinical validation of the assay showed 100% sensitivity and specificity for detecting ß-lactam resistance in 30 min by measuring hydrolysis of ceftriaxone (0.50 mg/mL) with the automated MBT STAR-BL software. Regarding the antibiotics evaluated, ceftriaxone yielded 70% more positive results than cefotaxime, 80% more than ceftazidime and 20% more than cefpodoxime, with 100% specificity. Cefepime revealed 100% sensitivity, but only 27% specificity. For the same incubation time, the automated software yielded on average 41% more positive results in relation to detecting resistance than qualitative interpretation of spectra. Conclusions: Our clinical validation of the method proved it to be highly reliable, simple to perform and time saving, transforming ß-lactam resistance detection by MALDI-TOF MS into a ready-to-use technique in clinical laboratories.

Disseminated aspergillosis in an immunocompetent patient with detectable bis(methylthio)gliotoxin and negative galactomannan / Aspergilosis diseminada en un paciente inmunocompetente con bis(metiltio)gliotoxina detectable y galactomanano negativo

Background. Disseminated invasive aspergillosis is an exceptional finding in immunocompetent hosts. As in immunocompromised patients, it has high mortality rates. Early diagnostic methods are required in order to properly manage the patient. Bis(methylthio)gliotoxin (bmGT) is a novel biomarker, useful in onco-hematological patients. Case report. A 70-year-old male, with non-insulin dependent type II diabetes mellitus and a past surgery history of aortic valve replacement with coronary by-pass five years ago, was seen in the emergency department with blurred vision. Three days later, endogen endophthalmitis was diagnosed in the ophthalmology clinic. During admission for the vitrectomy, he suffered an ischemia of the right lower limb. A thoracic computed tomography revealed a mycotic aneurysm of the ascending thoracic aorta and parietal thrombus. The ascending aorta was replaced and abundant brittle material of infectious appearance, found between the aortic valve graft and the aneurysm, was removed. Aspergillus fumigatus sensu stricto grew in both vitreous and aorta cultures. BmGT was detected in two serum samples obtained prior to intravenous antifungal treatment, which was then reduced after voriconazole treatment was started. Conclusions. Disseminated invasive aspergillosis is a severe disease regardless of the immune status of the patient. This case report suggests that bmGT could be a suitable early diagnostic biomarker, not only in neutropenic patients, but also in immunocompetent hosts (AU)

Antecedentes. La aspergilosis diseminada invasiva es un hallazgo excepcional en pacientes inmunocompetentes, y al igual que en los pacientes inmunodeficientes, alcanza valores de mortalidad elevados. Para el correcto manejo del paciente son necesarios métodos diagnósticos precoces. La bis(metiltio)gliotoxina es un nuevo biomarcador de gran utilidad en pacientes oncohematológicos. Caso clínico. Varón de 70 años de edad con diabetes mellitus tipo II no dependiente de insulina y antecedente de recambio valvular aórtico con by-pass coronario cinco años antes, que acude al Servicio de Urgencias por visión borrosa. Tres días después se le diagnosticó endoftalmitis endógena en la consulta de Oftalmología. Durante su ingreso para la vitrectomía presentó una isquemia del miembro inferior derecho. La tomografía computarizada de tórax reveló un aneurisma micótico en la aorta torácica ascendente y un trombo parietal. Se reemplazó la aorta ascendente y se eliminó abundante material friable de aspecto infeccioso entre la prótesis valvular aórtica y el aneurisma. En los cultivos de humor vítreo y aorta creció Aspergillus fumigatus sensu stricto. Se detectó bis(metiltio)gliotoxina en dos muestras de suero obtenidas antes del tratamiento antifúngico intravenoso, marcador que disminuyó tras comenzar el tratamiento con voriconazol. Conclusiones. La aspergilosis diseminada invasiva es una enfermedad grave independientemente del estado inmune del paciente. Este caso clínico evidencia que la bis(metiltio)gliotoxina podría ser un marcador diagnóstico precoz no solo en pacientes neutropénicos, sino también en huéspedes inmunocompetentes (AU)

Staphylococcus pseudintermedius Human Infection Cases in Spain: Dog-to-Human Transmission.

Staphylococcus pseudintermedius is an opportunistic pathogen that has been identified as infectious agent or colonizer mainly in dogs. S. pseudintermedius has been also detected in humans and more specifically in people in contact with dogs. In this study, the possible S. pseudintermedius pet-to-human transmission was analyzed in four clinical human cases. Two patients were dog owners and S. pseudintermedius was also detected as colonizer in these healthy animals. S. pseudintermedius isolates from patients and dogs of the same household showed identical pulsed-field gel electrophoresis patterns, sequence types (STs), and antimicrobial resistance phenotypes and genotypes, and were methicillin susceptible. Resistance to erythromycin, clindamycin, tetracycline, trimetoprim-sulfamethoxazole, and/or ciprofloxacin was identified among S. pseudintermedius strains. The lineages ST241 and the new ST521 were detected in the strains of the two dog-owner patients, respectively. The strains from the other two patients presented two new STs, ST719 and ST720. To our knowledge, this is the first description of human infections caused by S. pseudintermedius in Spain.

Disseminated aspergillosis in an immunocompetent patient with detectable bis(methylthio)gliotoxin and negative galactomannan.

BACKGROUND: Disseminated invasive aspergillosis is an exceptional finding in immunocompetent hosts. As in immunocompromised patients, it has high mortality rates. Early diagnostic methods are required in order to properly manage the patient. Bis(methylthio)gliotoxin (bmGT) is a novel biomarker, useful in onco-hematological patients. CASE REPORT: A 70-year-old male, with non-insulin dependent type II diabetes mellitus and a past surgery history of aortic valve replacement with coronary by-pass five years ago, was seen in the emergency department with blurred vision. Three days later, endogen endophthalmitis was diagnosed in the ophthalmology clinic. During admission for the vitrectomy, he suffered an ischemia of the right lower limb. A thoracic computed tomography revealed a mycotic aneurysm of the ascending thoracic aorta and parietal thrombus. The ascending aorta was replaced and abundant brittle material of infectious appearance, found between the aortic valve graft and the aneurysm, was removed. Aspergillus fumigatus sensu stricto grew in both vitreous and aorta cultures. BmGT was detected in two serum samples obtained prior to intravenous antifungal treatment, which was then reduced after voriconazole treatment was started. CONCLUSIONS: Disseminated invasive aspergillosis is a severe disease regardless of the immune status of the patient. This case report suggests that bmGT could be a suitable early diagnostic biomarker, not only in neutropenic patients, but also in immunocompetent hosts.

Evaluation of incubation time for dermatophytes cultures.

In general, it is recommended to incubate dermatophytes cultures for a minimum of 4 weeks. Several aspects of routine fungal cultures should be evaluated in order to implement appropriate and necessary changes. The aim of this study was to determine the optimum incubation time for routine dermatophytes cultures, analysing the time to find first fungal growth by visual observation. We recorded the time when the initial growth was detected for all dermatophyte isolates during a 4-year period. A total of 5459 dermatophyte cultures were submitted to our laboratory. From the total cultures, only 16 (1.42%) isolates were recovered over/after 17 days of incubation and only three dermatophyte species were recovered over 17 days. Fourteen isolates belong to Trichophyton rubrum, one isolate to Trichophyton mentagrophytes complex and one isolate to Epidermophyton floccosum. We concluded that an incubation period of 17 days is enough to establish a microbiological diagnosis of dermatophytosis.

A photobleaching resistant polymer supported hexanuclear molybdenum iodide cluster for photocatalytic oxygenations and photodynamic inactivation of Staphylococcus aureus.

The ability of a hexanuclear molybdenum cluster, [Mo6I8(CH3COO)6]2-, to carry out, upon irradiation, singlet oxygen mediated photocatalytic oxygenation reactions has been tested. This complex has been also supported on a solid polymeric matrix, yielding an immobilized photosensitizer with remarkable photostability and recyclability. Preliminary studies of antibacterial photoinactivation of Staphylococcus aureus have been conducted, with positive results.

In Vivo IS6110 Profile Changes in a Mycobacterium tuberculosis Strain as Determined by Tracking over 14 Years.

Transposition and homologous recombination of IS6110 appear in Mycobacterium tuberculosis along in vivo sequential infections. These events were checked in different clones of a successful strain, M. tuberculosis Zaragoza, with the focus on a variant in which integration of a copy of IS6110 in the origin of replication (oriC) region occurred.

Characterization of tetracycline and methicillin resistant Staphylococcus aureus strains in a Spanish hospital: is livestock-contact a risk factor in infections caused by MRSA CC398?

UNLABELLED: Tetracycline-resistance (Tet(R)) has been postulated as a marker of the livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage CC398. OBJECTIVES OF THE STUDY: to determine the spa-types and assigned MLST clonal complexes (CCs) among all 98 MRSA-Tet(R) strains recovered during 2011-2012 (from different patients) in a Spanish Hospital, analyzing the possible correlation with livestock-contact of the patients. All 98 strains were assigned to 9 CCs: CC398 (60.2%), CC1 (19.4%), CC5 (12.2%), and other CCs (8.2%). The 98 patients were classified into three groups: (A) contact with livestock-animals (n=25); (B) no-contact with livestock-animals (n=42); (C) no information about animal contact (n=31). A significant higher percentage of CC398 strains was obtained in group A (76%) than in group B (50%) (p<0.05), being the percentage in group C of 61.3%. Most of MRSA-Tet(R)-CC398 strains presented a multi-resistance phenotype, including erythromycin, clindamycin, and ciprofloxacin, and the most prevalent detected genes were tet(M) and erm(C). Three strains presented the phenotype macrolide-susceptibility/lincosamide-resistance and contained the vga(A) gene. MRSA-CC1 strains showed higher percentages of erythromycin/clindamycin resistance (95%/89%) than MRSA-CC398 strains (58%/63%), and this resistance was usually mediated by erm(C) gene. Most of MRSA-CC5 strains showed resistance to ciprofloxacin, tobramycin/kanamycin and erythromycin. None of the strains presented the genes lukF/lukS-PV, tsst-1, eta, etb or etd. All MRSA-CC398 strains lacked the genes of the immune-evasion-cluster, but MRSA-CC1 strains carried these genes (type E). In conclusion, although MRSA CC398 is detected in a significant higher proportion in patients with livestock-contact; its detection in people without this type of contact also indicates its capacity for human-to-human transmission.

Cutaneous sporotrichosis treated with photodynamic therapy: an in vitro and in vivo study.

BACKGROUND: Sporotrichosis is a fungal infection caused by Sporothrix schenckii complex, usually restricted to the skin, subcutaneous cellular tissue, and adjacent lymphatic vessels. Antimicrobial photodynamic therapy (aPDT) could be a good alternative to manage localized, superficial infections. CASE REPORT: A 65-year-old African woman was diagnosed with a fixed cutaneous sporotrichosis on her left arm, treated with itraconazol and oral terbinafine with partial improvement. Topical 16% methyl aminolevulinate (MAL, Metvix(®))-PDT was used without success. METHODS: An in vitro photoinactivation test with the isolated microorganism revealed phenothiazinium salts to be more effective than MAL. CONCLUSIONS: PDT with intralesional 1% methylene blue (MB) in combination with intermittent low doses of itraconazole obtained complete microbiological and clinical response.

[Kingella kingae pediatric septic arthritis]. / Artritis séptica pediátrica causada por Kingella kingae.

Kingella kingae is a bacterium that colonizes the upper respiratory tract. Despite its low pathogenicity in this location, previous respiratory pathological processes may favor its systemic spread causing bone and joint infections, mainly in children under five years. It can be considered an emerging pathogen in osteoarticular infection in pediatric patients. We report the case of a two-year-old girl with hips pain and limitation of both abduction and extension, and fever. Radiography and ultrasonography were compatible with transitory synovitis; showed scintigraphy inflammatory pathology of the right hip. Articular puncture was performed. The material showed altered biochemical parameters. Microbiological culture yielded isolation of a strain of K. kingae susceptible to beta-lactam antibiotics, azithromycin and trimethoprim-sulfamethoxazole. Blood cultures were negative. The patient was treated empirically with cloxacillin and cefotaxime iv. and continued with amoxicillin-clavulanate orally with osteoarticular improvement.

Artritis séptica pediátrica causada por Kingella kingae / Kingella kingae pediathric septic arthritis

Kingella kingae es un microorganismo que coloniza el tracto respiratorio superior. A pesar de su baja patogenicidad en esta localización, los procesos patológicos respiratorios previos pueden favorecer su diseminación sistémica y producir infecciones osteoarticulares, principalmente en menores de cinco años. En pacientes pediátricos, se considera un patógeno emergente en la infección osteoarticular. Presentamos el caso de una niña de dos años de edad con cuadro clínico de dolor de caderas, limitación de la abducción y extensión, y fiebre. La radiografía y la ecografía eran compatibles con sinovitis transitoria y la gammagrafía con patología osteoarticular infamatoria de la cadera derecha. Se realizó punción articular y los parámetros bioquímicos arrojaron resultados alterados. Se procesó microbiológicamente y se aisló K. kingae sensible a antibióticos betalactámicos, azitromicina y trimetoprima-sulfametoxazol. Los hemocultivos fueron negativos. Se trató empíricamente con cloxacilina y cefotaxima IV y se continuó oralmente con amoxicilina-clavulánico, con mejoría osteoarticular.

Kingella kingae is a bacterium that colonizes the upper respiratory tract. Despite its low pathogenicity in this location, previous respiratory pathological processes may favor its systemic spread causing bone and joint infections, mainly in children under five years. It can be considered an emerging pathogen in osteoarticular infection in pediatric patients. We report the case of a two-year-old girl with hips pain and limitation of both abduction and extension, and fever. Radiography and ultrasonography were compatible with transitory synovitis; showed scintigraphy infammatory pathology of the right hip. Articular puncture was performed. The material showed altered biochemical parameters. Microbiological culture yielded isolation of a strain of K. kingae susceptible to beta-lactam antibiotics, azithromycin and trimethoprim-sulfamethoxazole. Blood cultures were negative. The patient was treated empirically with cloxacillin and cefotaxime iv. and continued with amoxicillin-clavulanate orally with osteoarticular improvement.

Artritis séptica pediátrica causada por Kingella kingae / Kingella kingae pediathric septic arthritis

Kingella kingae es un microorganismo que coloniza el tracto respiratorio superior. A pesar de su baja patogenicidad en esta localización, los procesos patológicos respiratorios previos pueden favorecer su diseminación sistémica y producir infecciones osteoarticulares, principalmente en menores de cinco años. En pacientes pediátricos, se considera un patógeno emergente en la infección osteoarticular. Presentamos el caso de una niña de dos años de edad con cuadro clínico de dolor de caderas, limitación de la abducción y extensión, y fiebre. La radiografía y la ecografía eran compatibles con sinovitis transitoria y la gammagrafía con patología osteoarticular infamatoria de la cadera derecha. Se realizó punción articular y los parámetros bioquímicos arrojaron resultados alterados. Se procesó microbiológicamente y se aisló K. kingae sensible a antibióticos betalactámicos, azitromicina y trimetoprima-sulfametoxazol. Los hemocultivos fueron negativos. Se trató empíricamente con cloxacilina y cefotaxima IV y se continuó oralmente con amoxicilina-clavulánico, con mejoría osteoarticular.(AU)

Kingella kingae is a bacterium that colonizes the upper respiratory tract. Despite its low pathogenicity in this location, previous respiratory pathological processes may favor its systemic spread causing bone and joint infections, mainly in children under five years. It can be considered an emerging pathogen in osteoarticular infection in pediatric patients. We report the case of a two-year-old girl with hips pain and limitation of both abduction and extension, and fever. Radiography and ultrasonography were compatible with transitory synovitis; showed scintigraphy infammatory pathology of the right hip. Articular puncture was performed. The material showed altered biochemical parameters. Microbiological culture yielded isolation of a strain of K. kingae susceptible to beta-lactam antibiotics, azithromycin and trimethoprim-sulfamethoxazole. Blood cultures were negative. The patient was treated empirically with cloxacillin and cefotaxime iv. and continued with amoxicillin-clavulanate orally with osteoarticular improvement.(AU)

Identification of novel vga(A)-carrying plasmids and a Tn5406-like transposon in meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis of human and animal origin.

Nine staphylococcal strains of human and animal origin with a lincomycin-resistant/erythromycin-susceptible phenotype and carrying vga genes were characterised to determine the genetic elements involved in the dissemination of these uncommon resistance genes. These strains were typed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and/or spa typing. Antimicrobial susceptibility was studied by disk diffusion and agar dilution methods. Presence of the genes lnu(A), lnu(B), vga(A), vga(A)v, vga(B), vga(C), vga(E), lsa(B) and cfr was studied by PCR. Transformation experiments were carried out in all strains, and the plasmid or chromosomal gene location was determined by Southern blot analysis. Genetic environments of the vga genes were analysed by PCR mapping or inverse PCR and sequencing. Five meticillin-resistant Staphylococcus aureus (MRSA) ST398 strains and three Staphylococcus epidermidis strains harboured the gene vga(A), and one MRSA-ST8 strain contained the gene vga(A)v. One MRSA-ST398 strain, which also contained the gene lnu(A), showed the highest minimum inhibitory concentration (MIC) to lincomycin. The vga(A)v-positive strain presented lower MIC values than the vga(A)-positive strains. Presence of the pVGA plasmid was confirmed in two MRSA-ST398 strains. Four novel vga(A)-carrying plasmids were detected: pUR2355 (in two MRSA and one meticillin-susceptible S. epidermidis); pUR4128 (one MRSA); pUR3036 [one meticillin-resistant S. epidermidis (MRSE)]; and pUR3937 (one MRSE). The plasmid pUR4128 was very similar to pUR2355. Plasmids pUR3036 and pUR3937 were related and were very similar to plasmid pSE-12228-06. The gene vga(A)v was located in a transposon analogous to Tn5406. Therefore, four novel vga(A)-carrying plasmids and a variant of Tn5406 were identified in this study.

Acquisition of carbapenem resistance in multiresistant Klebsiella pneumoniae strains harbouring blaCTX-M-15, qnrS1 and aac(6')-Ib-cr genes.

Three closely related Klebsiella pneumoniae strains isolated from the same patient harboured bla(CTX-M-15), bla(OXA-1), bla(SHV-11), qnrS1, aac(6')-Ib-cr, oqxAB, aac(3)-II and aph(3')-Ia genes. Two of the isolates were recovered after treatment with meropenem and showed resistance to carbapenems. Sequencing of ompK35 and ompK36 porin genes of the carbapenem-resistant strains revealed the presence of premature stop codons in both, and OmpK35 and OmpK36 porins were not detected by SDS-PAGE. One carbepenem-resistant strain showed a high amount of LamB protein and did not express OmpK26 porin whereas the other strain expressed OmpK26 but not LamB. The lack of major porins apparently causes changes in the expression of other, specific, porins.

Long-term molecular surveillance of multidrug-resistant tuberculosis in Spain.

The data presented here span 11 years (1998-2008) of monitoring of multidrug-resistant tuberculosis (MDR-TB) clustering through molecular typing techniques in Spain. The molecular and epidemiological data of 480 multidrug-resistant Mycobacterium tuberculosis complex isolates were analyzed. Thirty-one clusters involving 157 (32.7%) patients were identified. The proportion of immigrants increased substantially over the study period reaching 65% in 2008; however, the clustering rate remained stable indicating that local transmission was little influenced by imported MDR-TB. The three major clusters respond to the persistence of two autochthonous strains throughout the study period and an extensively drug-resistant (XDR) Mycobacterium bovis outbreak with only two cases was reported since 2002. Molecular and epidemiological evidence for the importation of new strains and their spread within the community was found. Immigrant-only clusters most often grouped patients infected abroad with strains belonging to rare spoligotypes. Conversely, widespread spoligotypes of the Latin-American and Mediterranean (LAM) and Haarlem families were responsible for the majority of the MDR-TB local transmission. The demonstration of clusters spanning several Spanish regions that have been ongoing throughout the study period makes it advisable to maintain a continuous molecular surveillance in order to monitor the spread of MDR-TB.