[Show abstract][Hide abstract] ABSTRACT: Microbiological confirmation of a urinary tract infection (UTI) takes 24-48 h. In the meantime, patients are usually given empirical antibiotics, sometimes inappropriately. We assessed the feasibility of sequentially performing a Gram stain and MALDI-TOF MS mass spectrometry (MS) on urine samples to anticipate clinically useful information. In May-June 2012, we randomly selected 1000 urine samples from patients with suspected UTI. All were Gram stained and those yielding bacteria of a single morphotype were processed for MALDI-TOF MS. Our sequential algorithm was correlated with the standard semiquantitative urine culture result as follows: Match, the information provided was anticipative of culture result; Minor error, the information provided was partially anticipative of culture result; Major error, the information provided was incorrect, potentially leading to inappropriate changes in antimicrobial therapy. A positive culture was obtained in 242/1000 samples. The Gram stain revealed a single morphotype in 207 samples, which were subjected to MALDI-TOF MS. The diagnostic performance of the Gram stain was: sensitivity (Se) 81.3%, specificity (Sp) 93.2%, positive predictive value (PPV) 81.3%, negative predictive value (NPV) 93.2%, positive likelihood ratio (+LR) 11.91, negative likelihood ratio (-LR) 0.20 and accuracy 90.0% while that of MALDI-TOF MS was: Se 79.2%, Sp 73.5, +LR 2.99, -LR 0.28 and accuracy 78.3%. The use of both techniques provided information anticipative of the culture result in 82.7% of cases, information with minor errors in 13.4% and information with major errors in 3.9%. Results were available within 1 h. Our serial algorithm provided information that was consistent or showed minor errors for 96.1% of urine samples from patients with suspected UTI. The clinical impacts of this rapid UTI diagnosis strategy need to be assessed through indicators of adequacy of treatment such as a reduced time to appropriate empirical treatment or earlier withdrawal of unnecessary antibiotics.
PLoS ONE 01/2014; 9(1):e86915. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study evaluates MALDI-TOF MS capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following two methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%) we obtained a reliable identification by MALDI-TOF: in 103 isolates the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result) and in 18 isolates the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods
Diagnostic microbiology and infectious disease 01/2014; · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with inflammatory bowel disease (IBD) have an immune-deficient baseline status further modulated by immunosuppressive therapy that may promote the reactivation of latent viruses such as BK virus (BKV). The aim of this prospective study was to determine the prevalence of BKV infection in IBD patients and its potential relationship with the immunosuppressive treatment. Paired urine and plasma samples from 53 consecutive patients with IBD and 53 controls were analyzed. BKV detection was performed by conventional PCR and positive samples were further quantified by real-time PCR. No viremia was detected. BKV viruria was significantly more common in IBD patients than among the controls (54.7% versus 11.3%; P < 0.0001). The only risk factor for BKV viruria in IBD was age (47.2 ± 16.3 versus 37.8 ± 15.2; P = 0.036), and there was a trend towards higher rate of viruria in outpatients (61.5% versus 38.5%; P = 0.096) and in those not receiving ciprofloxacin (59.5% versus 40.5%; P = 0.17). A clear impact of the immunosuppressive regimen on BKV infection could not be demonstrated.
The Scientific World Journal 01/2014; 2014:970528. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introducción
La espectrometría de masas MALDI-TOF ha demostrado ser rápida y eficaz en la identificación de microorganismos que colonizan determinadas muestras clínicas. Nuestro objetivo fue analizar los valores de validez de la espectrometría de masas MALDI-TOF para predecir colonización y bacteriemia relacionada con el catéter (BRC) en todos los catéteres que llegaran al laboratorio de Microbiología.
Durante 3 meses, la espectrometría de masas MALDI-TOF se realizó sobre las puntas de catéter recibidas (previo rodamiento para cultivo). Las reglas de oro de colonización y BRC fueron, respectivamente, la presencia de ≥ 15 ufc/placa en el cultivo de la punta de catéter y el aislamiento del (de los) mismo(s) microorganismo(s) tanto en los hemocultivos como en el catéter colonizado (7 días antes o después de la retirada del catéter).
Se incluyeron un total de 182 catéteres intravasculares. La tasa global de colonización detectada por la técnica del rodamiento y la espectrometría de masas MALDI-TOF fue del 31,9 y del 32,4%, respectivamente. Hubo un total de 33 (18,1%) episodios de BRC. Los valores de validez de la espectrometría de masas MALDI-TOF para predecir colonización y BRC fueron, respectivamente: sensibilidad (69,0/66,7%), especificidad (84,7/75,2%), valor predictivo positivo (65,6/36,1%) y valor predictivo negativo (86,8/92,6%).
La espectrometría de masas MALDI-TOF puede ser una herramienta de diagnóstico alternativa para descartar BRC. Sin embargo, a pesar de haber demostrado ser más rápida que el cultivo convencional, son necesarios futuros estudios que mejoren el proceso pre-analítico.
Enfermedades Infecciosas y Microbiología Clínica 01/2014; · 1.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the last years, matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry has proved a rapid and reliable method for the identification of bacteria and yeast already isolated. The objective of this study was to evaluate this technology, as a routine method for the identification of microorganisms directly from grown blood culture bottles (BCB), before isolation, in a large collection of samples. For this purpose, 1,000 positive BCBs containing 1,085 microorganisms have been analyzed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF anticipated a species- or genus-level identification of 81.4% of the analyzed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1.000 analyzed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, versus 18 hours later when conventional identification methods are performed. Moreover, although further improvement of the sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in the Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causing pathogen within hours. This article is protected by copyright. All rights reserved.
Clinical Microbiology and Infection 11/2013; · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Influenza is easily overlooked in intensive care units (ICUs), particularly in patients with alternative causes of respiratory failure or in those who acquire influenza during their ICU stay. METHODS: We performed a prospective study of patients admitted to three adult ICUs of our hospital from December 2010 to February 2011. All tracheal aspirate (TA) samples sent to the microbiology department were systematically screened for influenza. We defined influenza as unsuspected if testing was not requested and the patient was not receiving empirical antiviral therapy after sample collection. RESULTS: We received TA samples from 105 patients. Influenza was detected in 31 patients and was classified as unsuspected in 15 (48.4%) patients, and as hospital acquired in 13 (42%) patients. Suspected and unsuspected cases were compared, and significant differences were found for age (53 versus 69 median years), severe respiratory failure (68.8% versus 20%), surgery (6.3% versus 60%), median days of ICU stay before diagnosis (1 versus 4), nosocomial infection (18.8% versus 66.7%), cough (93.8% versus 53.3%), localized infiltrate on chest radiograph (6.3% versus 40%), median days to antiviral treatment (2 versus 9), pneumonia (93.8% versus 53.3%), and acute respiratory distress syndrome (75% versus 26.7%). Multivariate analysis showed admission to the surgical ICU (odds ratio (OR), 37.1; 95% confidence interval (CI), 2.1 to 666.6; P = 0.01) and localized infiltrate on chest radiograph (OR, 27.8; 95% CI, 1.3 to 584.1; P = 0.03) to be independent risk factors for unsuspected influenza. Overall mortality at 30 days was 29%. ICU admission for severe respiratory failure was an independent risk factor for poor outcome. CONCLUSION: During the influenza season, almost one third of critical patients with suspected lower respiratory tract infection had influenza, and in 48.4%, the influenza was unsuspected. Lower respiratory samples from adult ICUs should be systematically screened for influenza during seasonal epidemics.
Critical care (London, England) 06/2012; 16(3):R104. · 4.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro studies have demonstrated that bluetongue virus (BTV)-induced vasoactive mediators could contribute to the endothelial cells dysfunction and increased vascular permeability responsible of lesions characteristic of bluetongue (BT) like oedema, haemorrhages and ischaemic necrosis in different tissues. However, few in vivo studies have been carried out to clarify the causes of these lesions. The aim of this study was to elucidate in vivo the pathogenetic mechanisms involved in the appearance of vascular lesions in different organs during BT. For this purpose, tissue samples from goats naturally infected with bluetongue virus serotype 1 (BTV-1) were taken for histopathological and immunohistochemical studies to determine the potential role of proinflammatory cytokines (tumour necrosis factor alpha, TNFα and interleukin one alpha, IL-1α) in the increased vascular permeability and their relationship with the presence of virus. Gross and histopathological examination revealed the presence of vascular damage leading to generalized oedema and haemorrhages. Immunohistochemical studies displayed that endothelial injury may have been due to the direct pathogenic effect of BTV infection on endothelial cells or may be a response to inflammatory mediators released by virus-infected endothelial cells and, possibly, other cell types such as monocytes/macrophages. These preliminary results of what appears to be the first in vivo study of tissue damage in small BT-infected ruminants suggest a direct link between the appearance of vascular changes and the presence of BTV-induced vasoactive cytokines.
Transboundary and Emerging Diseases 05/2012; · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To detect and monitor the sequential changes in virus levels, a reverse transcription quantitative real-time polymerase chain reaction assay using a TaqMan probe was carried out on frozen blood and tissues samples collected from calves experimentally infected with a non-cytopathic Bovine viral diarrhoea virus (BVDV) genotype 1 strain. Blood samples were collected among days 1-14 post-inoculation (p.i). On day 3 p.i, viral RNA was detected in blood samples from six of the eight inoculated animals. Viral RNA was detected in all remaining inoculated animals between 5 and 12 days p.i. The levels of viral RNA increased along the experiment, with a maximal peak between 6 and 9 days p.i. Analysis of virus load in tissues collected from calves euthanized on days 3, 6, 9 and 14 p.i displayed that BVDV was detected on day 3 p.i, being especially abundant in tonsils and ileocaecal valve, highlighting the role of tonsils as the main earliest viral replication sites as well as the principal source for virus spread to other lymphoid tissues and visceral organs. Coinciding with the highest viraemia levels, the highest viral loads were recorded at 9 days p.i. in tonsils, ileal lymph nodes, distal ileum and spleen, showing the main role of these secondary lymphoid organs in the pathogenic mechanisms of BVDV. However, virus levels in the liver and lung increased only towards the end of the infection. This fact could influence in the appearance of bovine respiratory diseases because of the capacity of BVDV for enhancing susceptibility to secondary infections.
Transboundary and Emerging Diseases 12/2011; 59(5):377-84. · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study is to evaluate the prevalence of BK virus (BKV) infection in HIV-positive patients receiving highly active antiretroviral therapy (HAART) in our hospital. The presence of BKV was analysed in urine and plasma samples from 78 non-selected HIV-infected patients. Clinical data were recorded using a pre-established protocol. We used a nested PCR to amplify a specific region of the BKV T-large antigen. Positive samples were quantified using real-time PCR. Mean CD4 count in HIV-infected patients was 472 cells/mm3 and median HIV viral load was <50 copies/mL. BKV viraemia was detected in only 1 HIV-positive patient, but 57.7% (45 out of 78) had BKV viruria, which was more common in patients with CD4 counts>500 cells/mm3 (74.3% vs 25.7%; p=0.007). Viruria was present in 21.7% of healthy controls (5 out of 23 samples, p=0.02). All viral loads were low (<100 copies/mL), and we could not find any association between BKV infection and renal or neurological manifestations. We provide an update on the prevalence of BKV in HIV-infected patients treated with HAART. BKV viruria was more common in HIV-infected patients; however, no role for BKV has been demonstrated in this population.
European Journal of Clinical Microbiology 11/2011; 31(7):1531-5. · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background Epidemiological surveys have revealed outbreaks of pandemic influenza A (H1N1) 2009 in several different contexts. Molecular characterization of the influenza virus could help to provide a more accurate description of these outbreaks.Objective To genotype pandemic influenza A (H1N1) 2009 isolates from an epidemiologically defined nosocomial outbreak.Study design We sequenced the neuraminidase (NA) and hemagglutinin (HA) influenza A (H1N1) 2009 genes from ten HIV-positive patients involved in an epidemiologically defined outbreak in the Clinical Microbiology and Infectious Diseases (CMID) Department. Sequences were aligned to search for specific genetic features of the involved strain. We also analyzed 37 unrelated influenza A (H1N1) 2009 cases from other hospital departments. All the sequences were used to obtain phylogenetic trees.Results Identical genotypic features were shared by nine of the 10 cases initially considered to be involved in the outbreak, but not by the remaining case. These features involved two silent mutations at N385 and V407 in the NA gene and three amino acid substitutions in the HA gene (D225E, A189T, and P300S). Searching for these substitutions in patients with influenza A (H1N1) 2009 hospitalized in other departments during the same period allowed us to identify an additional unsuspected immunocompetent case. The five outbreak-specific substitutions were absent in the remaining 36 unrelated controls. One of the substitutions (P300S) rendered detection of this variant by the CDC protocol inefficient. The other outbreak-specific substitutions (D225E and A189T) were identified at codons that have been analyzed in the context of virulence.Conclusions Genotyping is essential to ensure a more accurate description of pandemic influenza A (H1N1) 2009 outbreaks
Journal of Clinical Virology 10/2011; 52(2). · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological surveys have revealed outbreaks of pandemic influenza A (H1N1) 2009 in several different contexts. Molecular characterization of the influenza virus could help to provide a more accurate description of these outbreaks.
To genotype pandemic influenza A (H1N1) 2009 isolates from an epidemiologically defined nosocomial outbreak.
We sequenced the neuraminidase (NA) and hemagglutinin (HA) influenza A (H1N1) 2009 genes from ten HIV-positive patients involved in an epidemiologically defined outbreak in the Clinical Microbiology and Infectious Diseases (CMID) Department. Sequences were aligned to search for specific genetic features of the involved strain. We also analyzed 37 unrelated influenza A (H1N1) 2009 cases from other hospital departments. All the sequences were used to obtain phylogenetic trees.
Identical genotypic features were shared by nine of the 10 cases initially considered to be involved in the outbreak, but not by the remaining case. These features involved two silent mutations at N385 and V407 in the NA gene and three amino acid substitutions in the HA gene (D225E, A189T, and P300S). Searching for these substitutions in patients with influenza A (H1N1) 2009 hospitalized in other departments during the same period allowed us to identify an additional unsuspected immunocompetent case. The five outbreak-specific substitutions were absent in the remaining 36 unrelated controls. One of the substitutions (P300S) rendered detection of this variant by the CDC protocol inefficient. The other outbreak-specific substitutions (D225E and A189T) were identified at codons that have been analyzed in the context of virulence.
Genotyping is essential to ensure a more accurate description of pandemic influenza A (H1N1) 2009 outbreaks.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 08/2011; 52(2):129-32. · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BK virus (BKV) nephropathy is a common viral infection in renal transplant patients, with a prevalence of 1-9% at approximately 12 months after surgery. While it is widely agreed that reduction of immunosuppression should be the first intervention after diagnosis of BKV infection, there is no consensus on whether calcineurin inhibitors or antiproliferative drugs should be reduced first. Furthermore, target levels of immunosuppressive drugs are poorly defined, as are criteria for replacing one immunosuppressive agent with another. RESULTS: We report our series of 15 renal transplant patients who underwent surgery between September 2004 and March 2010 and who developed BKV infection. The first 8 patients were treated with reduction of immunosuppression; 7 of these patients received cidofovir and 6 received intravenous immunoglobulin. The remaining 7 renal transplant recipients received mammalian target of rapamycin inhibitors (imTOR). In this group, we observed faster and more efficacious BKV clearance in plasma and urine and a steady improvement in allograft function, with no episodes of acute allograft rejection during follow-up. The polymerase chain reaction assay for BKV in urine became positive in 2 patients in whom imTOR were stopped due to severe side effects. CONCLUSIONS: The use of imTOR should be considered a first step in the treatment of renal transplant recipients with BKV infection. In our experience, this change in treatment was safe and resulted in viral clearance.
[Show abstract][Hide abstract] ABSTRACT: Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines.
Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died.
There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.
PLoS ONE 01/2011; 6(10):e26666. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pandemic 2009 influenza A/H1N1 (H1N1v) is resistant to adamantanes, leaving neuraminidase inhibitors as the only therapeutic option. Other mutations are considered to be associated with virulence and clinical severity. However, out of the surveillance programs, few studies analyze the presence of resistance/virulent H1N1v variants in certain clinical circumstances.
To define the frequency and role of resistance and virulence mutations in a specific clinical circumstance-in patients with persistent infection by H1N1v.
Observational study of patients with persistent H1N1v infection admitted to our hospital.
NAI-resistance mutations were detected in 14.3% of cases with persistent infection (2/14), and in none of the non-persistent controls (0/15). These cases were initially infected with susceptible variants that acquired resistance at different time-points after therapy with oseltamivir (OTV). The first case (case 2) was an HIV-positive patient who rapidly acquired resistance 9 days after diagnosis (6 days on OTV) and whose infection resolved after standard OTV therapy. The second case (case 3) was a patient with chronic lymphocytic leukemia [corrected] and the longest viral persistence (59 days). The resistance mutation was detected in the specimen taken on day 37 after diagnosis (30 days on OTV). Once the resistance mutation was identified, OTV was substituted by zanamivir and the infection resolved. In addition to mutations encoding resistance, variants associated with virulence were also sought. The D225G mutation was not found in any case, whereas the D225E variant was identified in three persistent cases but also in two non-persistent ones. In one patient, the D225E substitution coincided with the H275 resistant mutation.
NAI-resistance mutations were detected, at rather different paces, in non-severe immunosuppressed cases with persistent infection by influenza A/H1N1v.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 11/2010; 50(2):114-8. · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the potential of red deer as bluetongue maintenance hosts and sentinels. Deer maintained detectable bluetongue virus (BTV) serotype 4 RNA for 1 year after the virus was cleared from livestock. However, the virus was not transmitted to yearlings. BTV serotype 1 RNA was detected in red deer immediately after its first detection in cattle.
[Show abstract][Hide abstract] ABSTRACT: The VP7 structural protein is the most abundant of the major core proteins and is highly conserved in all serotypes of bluetongue virus (BTV). The aim of this study was to develop immunohistochemical techniques for the detection of BTV VP7 in Bouin's- and formalin-fixed and paraffin wax-embedded tissues from small ruminants (sheep and goats) naturally infected with BTV. Tissue samples were taken from animals in which BTV infection had been confirmed by reverse transcriptase polymerase chain reaction. Optimal results were obtained by incubation of monoclonal antibody 2E9 on samples fixed with Bouin's solution or neutral buffered formalin. Optimum antigen retrieval for Bouin's-fixed samples was by microwave heating (6 min) of tissue samples in citrate buffer (pH 6.0, 0.01 M), while for formalin-fixed samples a 30 min heating period in pH 9.0 buffer was required. In both species, BTV was mainly detected in the spleen, lymph nodes and lungs; specifically within the arteriolar and capillary endothelial cells, together with macrophages and lymphocytes. The immunohistochemical method described will be a useful tool for future research.
Journal of comparative pathology 02/2010; 143(1):20-8. · 1.73 Impact Factor