Pneumococcal serotypes in children, clinical presentation and antimicrobial susceptibility in the PCV13 era.

The aim was to analyse invasive pneumococcal disease (IPD) serotypes in children aged ⩽17 years according to clinical presentation and antimicrobial susceptibility. We conducted a prospective study (January 2012-June 2016). IPD cases were diagnosed by culture and/or real-time polymerase chain reaction (PCR). Demographic, microbiological and clinical data were analysed. Associations were assessed using the odds ratio (OR) and 95% confidence intervals (CI). Of the 253 cases, 34.4% were aged <2 years, 38.7% 2-4 years and 26.9% 5-17 years. Over 64% were 13-valent pneumococcal conjugate vaccine (PCV13) serotypes. 48% of the cases were diagnosed only by real-time PCR. Serotypes 3 and 1 were associated with complicated pneumonia (P < 0.05) and non-PCV13 serotypes with meningitis (OR 7.32, 95% CI 2.33-22.99) and occult bacteraemia (OR 3.6, 95% CI 1.56-8.76). Serotype 19A was more frequent in children aged <2 years and serotypes 3 and 1 in children aged 2-4 years and 5-17 years, respectively. 36.1% of cases were not susceptible to penicillin and 16.4% were also non-susceptible to cefotaxime. Serotypes 14, 24F and 23B were associated with non-susceptibility to penicillin (P < 0.05) and serotypes 11, 14 and 19A to cefotaxime (P < 0.05). Serotype 19A showed resistance to penicillin (P = 0.002). In conclusion, PCV13 serotypes were most frequent in children aged ⩽17 years, mainly serotypes 3, 1 and 19A. Non-PCV13 serotypes were associated with meningitis and occult bacteraemia and PCV13 serotypes with pneumonia. Non-susceptibility to antibiotics of non-PCV13 serotypes should be monitored.

Infectious encephalitis: utility of a rational approach to aetiological diagnosis in daily clinical practice.

In this study we attempt to assess the utility of a simplified step-wise diagnostic algorithm to determinate the aetiology of encephalitis in daily clinical practice and to describe the main causes in our setting. This was a prospective cohort study of all consecutive cases of encephalitis in adult patients diagnosed between January 2010 and March 2015 at the University Hospital Vall d'Hebron in Barcelona, Spain. The aetiological study was carried out following the proposed step-wise algorithm. The proportion of aetiological diagnoses achieved in each step was analysed. Data from 97 patients with encephalitis were assessed. Following a simplified step-wise algorithm, a definite diagnosis was made in the first step in 53 patients (55 %) and in 12 additional cases (12 %) in the second step. Overall, a definite or probable aetiological diagnosis was achieved in 78 % of the cases. Herpes virus, L. monocytogenes and M. tuberculosis were the leading causative agents demonstrated, whereas less frequent aetiologies were observed, mainly in immunosuppressed patients. The overall related mortality was 13.4 %. According to our experience, the leading and treatable causes of encephalitis can be identified in a first diagnostic step with limited microbiological studies. L. monocytogenes treatment should be considered on arrival in some patients. Additional diagnostic effort should be made in immunosuppressed patients.

Epidemiology and Immediate Indirect Effects of Respiratory Viruses in Lung Transplant Recipients: A 5-Year Prospective Study.

The epidemiology of respiratory viruses (RVs) in lung transplant recipients (LTRs) and the relationship of RVs to lung function, acute rejection (AR) and opportunistic infections in these patients are not well known. We performed a prospective cohort study (2009-2014) by collecting nasopharyngeal swabs (NPSs) from asymptomatic LTRs during seasonal changes and from LTRs with upper respiratory tract infectious disease (URTID), lower respiratory tract infectious disease (LRTID) and AR. NPSs were analyzed by multiplex polymerase chain reaction. Overall, 1094 NPSs were collected from 98 patients with a 23.6% positivity rate and mean follow-up of 3.4 years (interquartile range 2.5-4.0 years). Approximately half of URTIDs (47 of 97, 48.5%) and tracheobronchitis cases (22 of 56, 39.3%) were caused by picornavirus, whereas pneumonia was caused mainly by paramyxovirus (four of nine, 44.4%) and influenza (two of nine, 22.2%). In LTRs with LRTID, lung function changed significantly at 1 mo (p = 0.03) and 3 mo (p = 0.04). In a nested case-control analysis, AR was associated with RVs (hazard ratio [HR] 6.54), Pseudomonas aeruginosa was associated with LRTID (HR 8.54), and cytomegalovirus (CMV) replication or disease was associated with URTID (HR 2.53) in the previous 3 mo. There was no association between RVs and Aspergillus spp. colonization or infection (HR 0.71). In conclusion, we documented a high incidence of RV infections in LTRs. LRTID produced significant lung function abnormalities. Associations were observed between AR and RVs, between P. aeruginosa colonization or infection and LRTID, and between CMV replication or disease and URTID.

La reacción en cadena de la polimerasa en el diagnóstico de la enfermedad meningocócica invasiva / The use of polymerase chain reaction in the diagnosis of invasive meningococcal disease

INTRODUCCIÓN OBJETIVOS: La enfermedad meningocócica invasiva (EMI) constituye un grave problema de salud pública. A pesar de que el cultivo es la técnica de referencia para su diagnóstico, la administración previa de antibiótico altera su sensibilidad. Los objetivos de este estudio son el análisis epidemiológico de la EMI en nuestro medio, evaluar la utilidad de la reacción en cadena de la polimerasa (PCR) para incrementar el diagnóstico de confirmación de la EMI y valorar la asociación de la administración de antibiótico con el resultado negativo del cultivo. Pacientes y métodos: Estudio retrospectivo de los pacientes menores de 16 a˜nos diagnosticados de EMI mediante cultivo, PCR o ambos, que ingresaron en nuestro centro en el periodo 2004- 2012. Resultados: Se incluyó a 75 pacientes, de los cuales el 52% presentó sepsis, el 30,7% meningitis y el 17,3% sepsis con meningitis. La PCR fue positiva en todas las muestras de sangre y líquido cefalorraquídeo analizadas, mientras que el cultivo tuvo una positividad muy inferior (50,7%). Recibieron antibiótico antes de la extracción de las muestras 40 pacientes (53,3%) y el 40% de ellos fueron confirmados por la PCR. Conclusiones: Gracias a la PCR se obtuvo un diagnóstico de confirmación de EMI en el 38,7% de los casos y del serogrupo, hecho relevante para la vigilancia epidemiológica y el estudio de la efectividad vacunal

INTRODUCTION AND OBJECTIVES AND AIMS: Invasive meningococcal disease (IMD) remains a serious public health problem. Although culture is the gold standard, previous antibiotic therapy reduces its sensibility. The aim of this study is the epidemiological analysis of IMD in our area, to assess the usefulness of polymerase chain reaction (PCR) to increase its diagnostic accuracy,and to show the association of antibiotic administration with the negative result of the culture. Patients and methods: A retrospective study was conducted on all children younger than 16 years with microbiologically (positive culture and/or PCR) confirmed IMD, admitted to our hospital between 2004-2012. Results: Seventy-five patients were included, of whom 52% had sepsis, 30.7% meningitis, and 17.3% with both of them. PCR was positive in all samples, whereas a positive was seen 50.7% of the cultures. Previously administered antibiotic was documented in 40 patients (53.3%), and 40% of them were confirmed by PCR only. Conclusions: PCR was the only test providing evidence for IMD diagnosis and serogroup determination in almost 39% of cases

[The use of polymerase chain reaction in the diagnosis of invasive meningococcal disease]. / La reacción en cadena de la polimerasa en el diagnóstico de la enfermedad meningocócica invasiva.

INTRODUCTION AND OBJECTIVES AND AIMS: Invasive meningococcal disease (IMD) remains a serious public health problem. Although culture is the gold standard, previous antibiotic therapy reduces its sensibility. The aim of this study is the epidemiological analysis of IMD in our area, to assess the usefulness of polymerase chain reaction (PCR) to increase its diagnostic accuracy,and to show the association of antibiotic administration with the negative result of the culture. PATIENTS AND METHODS: A retrospective study was conducted on all children younger than 16 years with microbiologically (positive culture and/or PCR) confirmed IMD, admitted to our hospital between 2004-2012. RESULTS: Seventy-five patients were included, of whom 52% had sepsis, 30.7% meningitis, and 17.3% with both of them. PCR was positive in all samples, whereas a positive was seen 50.7% of the cultures. Previously administered antibiotic was documented in 40 patients (53.3%), and 40% of them were confirmed by PCR only. CONCLUSIONS: PCR was the only test providing evidence for IMD diagnosis and serogroup determination in almost 39% of cases.

Serotype 3 is a common serotype causing invasive pneumococcal disease in children less than 5 years old, as identified by real-time PCR.

Serotype 3 is one of the most often detected pneumococcal serotypes in adults and it is associated with serious disease. In contrast, the isolation of serotype 3 by bacterial culture is unusual in children with invasive pneumococcal disease (IPD). The purpose of this study was to learn the serotype distribution of IPD, including culture-negative episodes, by using molecular methods in normal sterile samples. We studied all children<5 years of age with IPD admitted to two paediatric hospitals in Catalonia, Spain, from 2007 to 2009. A sequential real-time polymerase chain reaction (PCR) approach was added to routine methods for the detection and serotyping of pneumococcal infection. Among 257 episodes (219 pneumonia, 27 meningitis, six bacteraemia and five others), 33.5% were identified by culture and the rest, 66.5%, were detected exclusively by real-time PCR. The most common serotypes detected by culture were serotypes 1 (26.7%) and 19A (25.6%), and by real-time PCR, serotypes 1 (19.8%) and 3 (18.1%). Theoretical coverage rates by the PCV7, PCV10 and PCV13 vaccines were 10.5, 52.3 and 87.2%, respectively, for those episodes identified by culture, compared to 5.3, 31.6 and 60.2% for those identified only by real-time PCR. Multiplex real-time PCR has been shown to be useful for surveillance studies of IPD. Serotype 3 is underdiagnosed by culture and is important in paediatric IPD.

Characterization of plasma protein activity in riboflavin and UV light-treated fresh frozen plasma during 2 years of storage at -30 degrees C.

BACKGROUND: The Mirasol Pathogen Reduction Technology System (PRT) for Plasma (CaridianBCT) is based on a riboflavin and UV light treatment process resulting in pathogen inactivation due to irreversible, photochemically induced damage of nucleic acids. This study evaluated the in vitro protein quality of plasma products treated with riboflavin and UV light following treatment and subsequent storage for up to 104 weeks at -30 degrees C. MATERIALS AND METHODS: Apheresis and whole blood-derived plasma products were combined with riboflavin solution and exposed to ultraviolet light. Treated plasma was then flash frozen, within 8 h of collection, stored at -30 degrees C for up to 104 weeks and analysed at different stages of storage using standard coagulation assays. Results were compared with paired, untreated units stored for the same intervals. RESULTS: The average percent protein retention for all time-points in PRT-treated plasma samples after 36, 69, 87 and 104 weeks of storage at -30 degrees C in comparison with controls held under similar conditions were: Total Protein, 101%, Factor VIII, 79%, Fibrinogen, 78%, Factor II, 87%, Factor XII, 86%, Factor X, 84% and Factor IX, 81%. Anticoagulant and inhibitor proteins showed between 90% and 100% retention after 1 year (52 weeks) and 69 weeks of storage. No clinically relevant complement activation was observed in treated and stored samples. CONCLUSION: Riboflavin and UV light-treated plasma demonstrates reductions in several plasma coagulation factors following treatment. This reduction in activity levels is noted immediately after treatment and remains relatively constant during 2 years of storage at -30 degrees C.

Impact of valganciclovir on Epstein-Barr Virus polymerase chain reaction in pediatric liver transplantation: preliminary report.

UNLABELLED: Preemptive therapy with ganciclovir has been recommended in the pediatric liver transplant strategy to avoid the development of posttransplant lymphoproliferative disorder (PTLD) from an high Epstein-Barr virus (EBV) is detected. We sought viral load to analyze the response to preemptive therapy with valganciclovir (VGC) in children with liver transplantations and an high quantitative EBV-PCR. METHODS: From June 2005 to December 2007, we tested 979 EBV-PCR among 80 pediatric liver transplant recipients, from those 21/80 PCR were tested from the date of transplantation and 59/80 belonged to the historical cohort (7/59 had a prior history of PTLD). Patients were divided into 2 groups depending upon whether they did (n = 22) or did not (n = 19) receive VGC treatment. The response to VGC was considered complete, if the PCR was negative at 30 and 60 days of treatment; and partial, when the PCR decreased at least 50%. Ganciclovir blood levels tested in 109 cases instances and correlated with the EBV-PCR. RESULTS: A total of 369 (33%) positive PCR were detected in 36/80 patients (mean, 75,000 copies; range = 5000-4,200,000). Among the 22 episodes treated for 30 days, 34% showed complete responses, 41%, partial, and 23%, no response. Among the non-treated group the rates were 6%, 25%, and 68%, respectively (P = .01). However, no differences were observed among those episodes treated for 60 days. At the administered doses, hardly any patient reached the recommended ganciclovir therapeutic level at 2 hours (6 micro/mL). However, the mean PCR was lower when the ganciclovir levels were greater than 4 mg/L when compared with lower levels (P = .03). CONCLUSION: After 30 days of treatment there was a response to VGC in the EBV viral load. There was high interpatient variability of ganciclovir serum concentrations, suggesting the need for pharmacokinetic monitoring to optimize treatment. There was a relationship between the concentration of ganciclovir and the EBV viral load.

The immune response against herpesvirus is more prominent in the early stages of MS.

OBJECTIVE: To assess antibody levels against human herpesvirus type 6 (HHV-6), Epstein-Barr virus (EBV), and Chlamydia pneumoniae (CP) in serum from individuals in the early and late phase of MS. RESULTS: A strong association was found between anti-HHV-6 immunoglobulin M antibodies and early MS (clinically isolated syndromes at high risk for MS, and short duration active relapsing-remitting MS) when compared with healthy controls and secondary progressive MS. Moreover, in this group of patients, titers for anti-EBV immunoglobulin G were also elevated. The authors found no association between the levels of serum antibodies against CP and MS, nor did they detect the presence of DNA for these pathogens in the serum of patients with MS. Finally, serum from two patients with other inflammatory neurologic diseases also had elevated immunoglobulin M antibodies to HHV-6, indicating that the presence of this antibody is not specific to MS. CONCLUSION: An immune response against herpesviruses such as HHV-6 and EBV is associated with early MS.

Isolation of mycobacteria from frozen fish destined for human consumption.

Mycobacteria were isolated from defrost water and tissue of sole (Solea solea), hake (Merluccius merluccius), cod (Gadus morhua), ling (Genypterus blacodes), and monkfish (Lophius piscatorius) on Löwenstein-Jensen medium after incubation at different temperatures. Samples of frozen fish were obtained under sterile conditions inside a refrigeration chamber (-18 to -22 degrees C) in a wholesale market from which these products are distributed to shops for retail sale and human consumption.

Heat shock results in cell cycle delay and synchronisation of mitotic domains in cellularised Drosophila melanogaster embryos.

Cells of Drosophila embryos that are subjected to a 37 degrees C temperature shock whilst undergoing the S-phase of cell cycle 14 arrest with their microtubules in an interphase-like state, and with nuclei showing unusual chromatin condensation. They do not recover from this state within a 30 minute period even though extensive gastrulation movements can occur. Cells of embryos heat shocked in G2-phase are delayed in interphase with high levels of cyclins A and B. Within ten minutes recovery from heat shock, cells enter mitosis throughout the embryo. The degradation of the mitotic cyclins A and B in these synchronised mitotic domains does not follow the normal timing, but is delayed. These findings point to a need for caution when interpreting experiments that use the heat shock promoter to study the expression of cell cycle control genes in Drosophila.

[Evaluation of various criteria for the interpretation of western blot for the diagnosis of human immunodeficiency virus infection. Spanish Group for the Study of HIV-2]. / Evaluación de diferentes criterios de interpretación del Western blot para el diagnóstico de la infección por el virus de la inmunodeficiencia humana. Grupo Español para el Estudio del VIH-2.

BACKGROUND: The Western blot (WB) is the most commonly used test to confirm the presence of antibodies against the human immunodeficiency virus type 1 (HIV-1). Different criteria of interpretation of the band profile have been proposed with there being no unanimity as to its reliability. The sensitivity and specificity of several criteria proposed for the interpretation of WB were evaluated and the individual significance of the reactivity of each band of the WB was analyzed. METHODS: The presence of antibodies against HIV-1 was prospectively studied in 8,073 samples of subjects with risk of infection. A total of 1,993 (25%) were reactive by ELISA and 1,261 were analyzed by WB, with a semiquantitative reading of the bands with a point scale from 0 to 2 being performed. The final interpretation of the WB (negative, doubtful, or positive) was carried out following 5 recommendations of usage. A test designed with synthetic peptides (Pepti-lav) was used as a reference and in discordant cases, other more specific serologic tests and/or genetic analysis by polymerase chain reaction (PCR) were performed. RESULTS: In order of frequency, the greater sensitivity was found to be for the CRSS (Consortium for Retrovirus Serology Standardization) criteria (97.9%), OMS (96.6%), CDC (Center for Disease Control) (95.9%), ARC (American Red Cross) (95.6%) and FDA (99.8%). The greatest specificity was for the criteria of the OMS, and FDA (99.8%). In order of frequency, the most frequent bands in HIV-1 + individuals were gp160 (99%), gp120, p24, p31, p55, p68, gp41, and p17 (68%). In non infected individuals, the recognized bands were, in decreasing order, p24, p17, p55, p68, p31, and glucoproteins. CONCLUSIONS: Different criteria of interpretation of the Western blot provide different degrees of sensitivity and specificity. The Western blot is a non standardized, expensive, laborious technique of subjective interpretation which provides an appreciable number of undetermined results.

[A multicenter study of infection with human immunodeficiency virus type-2 (HIV-2) in Spain (1991). The Spanish Group for the Study of HIV-2]. / Estudio multicéntrico de la infección por el virus de la inmunodeficiencia humana tipo 2 (VIH-2) en España (1991). Grupo Español para el Estudio del VIH-2.

BACKGROUND: To January 1991 thirteen cases of HIV-2 infection had been reported in Spain. Paradoxically, neighboring countries, i.e. France and Portugal, have reported more than one thousand cases, and are the most HIV-2 prevalent areas outside West Africa. We report the results of a prospective, nationwide study on the prevalence of HIV-2 infection conducted in Spain in 1991. In addition, an evaluation of testing methodologies is made. METHODS: Sera collected from 8,073 individuals at high-risk for HIV infection were screened by a combined HIV-1 plus HIV-2 ELISA. Reactive samples were further evaluated by three tests, as HIV-1 Western blot (WB), HIV-2 specific WB, and a synthetic peptide assay immuno-dot (Pepti-lav, Pasteur). RESULTS: Fifteen (0.18%) individuals met criteria of HIV-2 infection in both specific WB and SPA. Four (27%) of them showed reactivity to both HIV-1 and HIV-2, and the dual infection was confirmed by polymerase chain reaction (PCR) in 2 out of 3 available samples. The SPA showed higher sensitivity and specificity than WB in the diagnosis and distinction of HIV-1 and HIV-2 infections. CONCLUSIONS: To January 1992, 28 cases of HIV-2 infection have been described in Spain. All but two were African immigrants. The first cases of HIV-1 plus HIV-2 coinfection have been identified. In HIV high-risk populations, SPA may provide an excellent alternative to WB to confirm ELISA reactive samples.

[DNA amplification using PCR in the diagnosis of tuberculosis]. / Amplificación de ADN mediante RCP en el diagnóstico de la tuberculosis.

BACKGROUND: To evaluate the DNA amplification technique of M. tuberculosis with PCR in clinical samples. METHODS: We have studied 57 clinical samples of 49 patients using PCR and culture in conventional media. We evaluate the final diagnosis of tuberculosis depending upon the bacteriologic results of all available samples. RESULTS: We reached a 58% sensitivity and 100% specificity. In 9 samples with positive PCR the culture was negative. The samples belonged to 9 patients with tuberculosis (4 with active disease and 5 with past microbiological diagnosis). In 32 samples with negative PCR test, the microorganism was isolated in 9 cases. SUMMARY: More studies are needed before PCR technique could be recommended for widespread use in the diagnosis of tuberculosis. Its long duration, the equipment needed and the false negative results are among its limitations.

Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos.

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.

The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts.

The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

We show that the sequence of Drosophila cyclin B has greater identity with B-type cyclins from other animal phyla than with Drosophila cyclin A, suggesting that the two cyclins have distinct roles that have been maintained in evolution. Cyclin A is not detectable in unfertilized eggs and is present at low levels prior to cellularization of the syncytial embryo. In contrast, the levels of cyclin B remain uniformly high throughout these developmental stages. In cells within cellularized embryos and the larval brain, cyclin A accumulates to peak levels in prophase and is degraded throughout the period in which chromosomes are becoming aligned on the metaphase plate. The degradation of cyclin B, on the other hand, does not occur until the metaphase-anaphase transition. In cells arrested at c-metaphase by treating with microtubule destabilizing drugs to prevent spindle formation, cyclin A has been degraded in the arrested cells, whereas cyclin B is maintained at high levels. These observations suggest that cyclin A has a role in the G2-M transition that is independent of spindle formation, and that entry into anaphase is a key requirement for the degradation of cyclin B.