Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing.

OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Bacterial evolution: bacteria play pass the gene.

DNA transfer between related bacterial species is enhanced by species-specific uptake sequences. These sequences have been used to identify genes that have been transferred from Haemophilus to Neisseria, providing a clear example of interspecific transfer of DNA in the evolution of the pathogenic Neisseria.

Implications of sequencing bacterial genomes for pathogenesis and vaccine development.

Improvements in homology search methodology and functional predictions are being complemented by the increase in the volume of sequence data with which comparative analyses can be performed. The experimental methods needed for investigation of gene function and expression in a variety of model systems of infection continue to develop. The identification of surface-exposed microbial structures and their conservation in natural populations of pathogenic species offers prospects for developing novel vaccines. A major challenge is the development of efficient screening methods to select the most promising candidates, such as immunisation with DNA.

Hemostasis and cold knife cone biopsy: a prospective randomized trial comparing a suture versus non-suture technique.

Two methods of obtaining hemostasis after cold knife cone biopsy were compared in a prospective randomized trial involving 200 patients. One method relied primarily on hemostatic sutures, and the other involved the use of a styptic solution (Monsel's solution) and vaginal pack, thus avoiding the use of sutures altogether. The short- and long-term morbidity in these two groups were compared and 12-month follow-up was completed. The use of sutures did not reduce the incidence of primary hemorrhage. Secondary hemorrhage was twice as frequent in the suture group, although this trend did not quite reach statistical significance. During long-term follow-up, significantly more patients in the suture group developed menstrual symptoms, cervical stenosis, and unsatisfactory colposcopy, requiring further operative intervention as a result.

A study of the interaction between recombinant bactericidal permeability increasing protein (rBPI(23)) and gentamicin.

rBPI(23) is a recombinant protein based upon the N-terminal sequence of bactericidal permeability increasing protein (BPI), a protein present in the granules of polymorphonuclear leukocytes. BPI has antibacterial activity against Gram-negative organisms and potent endotoxin neutralising properties. rBPI(23) has been developed as a potential agent for use in Gram-negative sepsis and has completed phase 1 clinical trials. In this study a broth microdilution chequerboard method has been used to investigate the interaction between rBPI(23) and gentamicin, an antibiotic used in a similar clinical setting. Using organisms with a range of inherent susceptibilities to rBPI(23), additive or synergistic effects were seen which tended to be proportional to the sensitivity to rBPI(23) alone.

Genome sequencing and annotation.

The availability of complete microbial genome sequences enormously facilitates experimental molecular investigations of the respective organisms by providing complete lists of genes, their genetic contexts, and their predicted functions. This can be used in a number of ways to focus studies on bacterial pathogenesis and also vaccine development (1,2). The complete genome sequences from two unrelated strains of Neisseria meningitidis, a derivative of isolate MC58 which originally expressed serogroup B capsule and strain Z2491, which is serogroup A, are now available (3,4). The genome sequences of both these strains were determined using the whole genome shotgun approach (5). In this approach, randomly sheared chromosomal DNA is cloned to make a small insert library (1.5-2.0 kb for MC58, 0.5-0.8 kb and 1.0-1.5 kb for Z2491), then each insert is sequenced from both ends using plasmidspecific primers. For the MC58 genome sequence, a large insert lambda library (8-24 kb) was also used. In the initial sequencing phase, 6-8 times coverage of the estimated size of the genome is generally achieved. The DNA sequences are linked together (assembled) into large contigs (a derivative of the word contiguous). Polymerase chain reaction (PCR) and sequencing of large insert libraries are then used to join the contigs, close gaps, and resolve ambiguities (see ref. 6 for a review).

Risk factors for major obstetric haemorrhage.

The factors associated with major obstetric haemorrhage were analyzed using data relating to 37,497 women delivered in 1988 in National Health Service maternity units in the North West Thames Region, UK. Four hundred ninety-eight cases (1.33%) were complicated by haemorrhage of 1000 ml or more. Intrinsic factors associated with significant risk ratios (99% confidence intervals) included placental abruption 12.6 (7.61-20.9), placenta praevia 13.1 (7.47-23.0), multiple pregnancy 4.46 (3.01-6.61) and obesity 1.64 (1.24-2.17), but not high parity. Significant risk factors related to obstetric management and delivery included retained placenta 5.15 (3.36-7.87), induced labour 2.22 (1.67-2.96), episiotomy 2.06 (1.36-3.11) and birthweight 4 kg or more 1.90 (1.38 to 2.60). Among the 59 women who lost 1000 ml or more in association with a spontaneous vaginal delivery with an intact perineum, significant risk ratios (99% confidence intervals) were retained placenta 13.7 (5.92-31.8) and induced labour 2.35 (1.11-4.98). These data provide a more comprehensive assessment of risk factors for potentially life threatening haemorrhage in British obstetric practice than is possible using maternal mortality statistics. The hazards of well known factors such as multiple pregnancy, abruption, placenta praevia and caesarean delivery were confirmed but attention is drawn to the potential risk of haemorrhage associated with obesity or a large baby and to that associated with retained placenta in women classified as 'low risk'.

Mode of delivery after one caesarean section: audit of current practice in a health region.

OBJECTIVE: To audit the subsequent obstetric management of women who had had one previous baby delivered by caesarean section. DESIGN: Retrospective analysis of a regional obstetric database. SETTING: Data derived from the 17 obstetric units in North West Thames region. SUBJECTS: 1059 women who delivered a singleton fetus of at least 37 weeks' gestation with a cephalic presentation in 1988 who had a history of one previous caesarean section and no other deliveries. MAIN OUTCOME MEASURES: Mode of delivery, postnatal morbidity, and duration of hospital stay. RESULTS: 395 (37%) women were delivered by elective repeat caesarean section and 664 (63%) were allowed a trial of labour. Maternal height and birth weight of the previous infant differed significantly between those who were and those who were not allowed to labour. 471 (71%) of those allowed to labour achieved a vaginal delivery. In individual units there was no significant correlation between the proportion of patients allowed to labour and the rate of the successful trial of labour. There was a trend towards greater success rates in units that allowed a longer duration of labour (p less than 0.05) and units with greater use of oxytocin for augmentation of labour (not significant). Both elective and intrapartum caesarean section was associated with a significantly higher rate of postnatal infection than vaginal delivery (14.7% and 16.0% v 3.4%). CONCLUSIONS: In patients with a history of caesarean section there is no evidence that the likelihood of successful vaginal delivery after trial of labour is modified by the proportion of such patients allowed the option of attempted vaginal delivery. Until selection criteria of adequate prognostic value can be identified a more liberal approach to allowing women a trial of labour seems justified.

Outcome of breech delivery at term.

OBJECTIVE: To compare neonatal mortality and morbidity in term infants presenting by the breech and delivered vaginally or by caesarean section. DESIGN: Population based comparison of outcomes. Data derived from the St Mary's maternity information system. SETTING: North West Thames Regional Health Authority, 1988-90. SUBJECTS: 3447 singleton fetuses presenting by the breech at term. MAIN OUTCOME MEASURES: Intrapartum and neonatal mortality, low Apgar scores, intubation at birth, and admission to special care baby units. RESULTS: After the exclusion of babies with congenital anomalies the incidence of intrapartum and neonatal death associated with vaginal birth was 8/961 (0.83%) compared with 1/2486 (0.03%) in babies born by caesarean section (relative risk 20, 95% confidence interval 2.5 to 163). The numbers of low Apgar scores and neonatal intubation were doubled in babies born vaginally or by emergency caesarean section compared with those delivered by elective operation. CONCLUSIONS: The good neonatal outcome associated with elective caesarean delivery of the term breech fetus may influence the decision of women and their obstetricians about mode of delivery.

Oxytocin infusion during second stage of labour in primiparous women using epidural analgesia: a randomised double blind placebo controlled trial.

OBJECTIVE: To determine whether the high rate of forceps delivery associated with the use of epidural analgesia could be reduced through giving an intravenous infusion of oxytocin during the second stage of labour. DESIGN: A randomised, double blind, placebo controlled trial. SETTING: Delivery suites in three hospitals. SUBJECTS: 226 Primiparous women with adequate epidural analgesia in whom full dilatation of the cervix had been achieved without prior stimulation with oxytocin. INTERVENTION: An infusion of oxytocin or placebo starting at the diagnosis of full cervical dilatation at an initial dose rate of 2 mU/min increasing to a maximum of 16 mU/min. MAIN OUTCOME MEASURES: The outcome of labour was assessed in terms of the duration of the second stage, mode of delivery, fetal condition at birth, postpartum blood loss, and the incidence of perineal trauma. RESULTS: Treatment with oxytocin was associated with a shorter second stage (p = 0.01), a reduction in the number of non-rotational forceps deliveries (p = 0.03), and less perineal trauma (p = 0.03) but was not associated with any reduction in the number of rotational forceps deliveries performed for malposition of the occiput. No adverse effects on fetal condition at birth or in the early puerperium were seen in association with the use of oxytocin. CONCLUSIONS: The use of an oxytocin infusion may reduce the high rate of operative delivery associated with epidural analgesia provided that the fetal occiput is in an anterior position at the onset of the second stage of labour but within the dose range studied does not seem to correct malposition of the fetal occiput.

An oncogenic role of eIF3e/INT6 in human breast cancer.

Altered expression of the eukaryotic translation initiation factor 3 (eIF3) subunit eIF3e/INT6 has been described in various types of human cancer, but the nature of its involvement in tumorigenesis is not yet clear. Using immunohistochemical analysis of 81 primary breast cancers, we found that high tumor grade correlated significantly with elevated cytoplasmic eIF3e level in epithelial tumor cells. Analysis of protein synthesis after siRNA-mediated knockdown in breast cancer cell lines indicated that eIF3e is not required for bulk translation. Microarray analysis of total and polysomal RNAs nonetheless identified distinct sets of mRNAs regulated either positively or negatively by eIF3e; functional classification of these revealed a marked enrichment of genes involved in cell proliferation, invasion and apoptosis. Validated mRNA targets regulated positively at the translational level by eIF3e included urokinase-type plasminogen activator and apoptotic regulator BCL-XL, whereas synthesis of proteins including the mitotic checkpoint component MAD2L1 was negatively regulated. Finally, eIF3e-depleted breast carcinoma cells showed reduced in vitro invasion and proliferation. Taken together, our study data suggest that eIF3e has a positive role in breast cancer progression. It regulates the translation, and in some cases abundance, of mRNAs involved in key aspects of cancer cell biology.

Why monitor peak vancomycin concentrations?

Peak and trough serum concentrations are routinely measured to monitor vancomycin therapy. Optimal therapy depends upon maintaining a concentration above that necessary for antibacterial activity and is therefore determined by the trough concentration. I determined the post dose increases in serum drug concentrations in routine clinical practice in adult patients without renal failure. Mean increases of 16.6 mg/L (SD 6.1) (peak samples collected 1 h post infusion) were measured from 165 paired samples. The results suggest that as long as trough concentrations do not exceed 15 mg/L, peak levels will not exceed normally accepted safe concentrations, and therefore do not need to be measured.

Prolactinoma during pregnancy causing compression symptoms responding to bromocriptine therapy.

A woman with pituitary macroadenoma causing pressure symptoms and a partial right third cranial nerve palsy during pregnancy is described. Complete resolution occurred using oral bromocriptine therapy alone and the remainder of the pregnancy was uneventful.

Controversies: the mature breech should be delivered by elective cesarean section.

The fetal hazards of vaginal breech delivery have been recognised for centuries. In recent years the increased safety of Cesarean delivery has prompted a steady rise in its use for the delivery of the term breech fetus. In many maternity units more than 90% of breech babies are delivered in this way. In contrast some obstetricians argue that with appropriate selection criteria, safe, selective, vaginal breech delivery is possible. This argument is not supported by population studies, which indicate that perinatal mortality and morbidity rates associated with attempts at vaginal breech delivery are between 1-2%.

Closed suction wound drainage and lower-segment caesarean section.

In a randomized controlled study of wound suction drainage after transverse suprapubic incision for lower-segment caesarean section no significant advantages could be demonstrated for routine drainage in terms of wound infection, haematoma formation, duration of hospital stay or analgesic requirements.