Interventions in the management of infection in the foot in diabetes: a systematic review.

The expert panel on diabetic foot infection (DFI) of the International Working Group on the Diabetic Foot conducted a systematic review seeking all published reports relating to any type of treatment for infection of the foot in persons with diabetes published as of 30 June 2014. This review, conducted with both PubMed and EMBASE, was used to update an earlier one undertaken on 30 June 2010 using the same search string. Eligible publications included those that had outcome measures reported for both a treated and a control population that were managed either at the same time, or as part of a before-and-after case design. We did not include studies that contained only information related to definition or diagnosis, but not treatment, of DFI. The current search identified just seven new articles meeting our criteria that were published since the 33 identified with the previous search, making a total of 40 articles from the world literature. The identified articles included 37 randomised controlled trials (RCTs) and three cohort studies with concurrent controls, and included studies on the use of surgical procedures, topical antiseptics, negative pressure wound therapy and hyperbaric oxygen. Among the studies were 15 RCTs that compared outcomes of treatment with new antibiotic preparations compared with a conventional therapy in the management of skin and soft tissue infection. In addition, 10 RCTs and 1 cohort study compared different treatments for osteomyelitis in the diabetic foot. Results of comparisons of different antibiotic regimens generally demonstrated that newly introduced antibiotic regimens appeared to be as effective as conventional therapy (and also more cost-effective in one study), but one study failed to demonstrate non-inferiority of a new antibiotic compared with that of a standard agent. Overall, the available literature was both limited in both the number of studies and the quality of their design. Thus, our systematic review revealed little evidence upon which to make recommendations for treatment of DFIs. There is a great need for further well-designed trials that will provide robust data upon which to make decisions about the most appropriate treatment of both skin and soft tissue infection and osteomyelitis in diabetic patients.

Genomic analysis of extra-intestinal pathogenic Escherichia coli urosepsis.

Urosepsis is a bacteraemia infection caused by an organism previously causing an infection in the urinary tract of a patient, a diagnosis which has been classically confirmed by culture of the same species of bacteria from both blood and urine samples. Given the new insights afforded by sequencing technologies into the complicated population structures of infectious agents affecting humans, we sought to investigate urosepsis by comparing the genome sequences of blood and urine isolates of Escherichia coli from five patients with urosepsis. The results confirm the classical urosepsis hypothesis in four of the five cases, but also show the complex nature of extra-intestinal E. coli infection in the fifth case, where three distinct strains caused two distinct infections. Additionally, we show there is little to no variation in the bacterial genome as it progressed from urine to blood, and also present a minimal set of virulence genes required for bacteraemia in E. coli based on gene association. These suggest that most E. coli have the genetic propensity to cause bacteraemia.

Molecular epidemiology and genetic diversity of pneumococcal carriage among children in Beni State, Bolivia.

During 2007, a study of pneumococcal carriage in children was performed in two towns (Trinidad and Riberalta) in the Beni region of the Bolivian Amazon basin. Little has previously been reported regarding the epidemiology of pneumococcal carriage in Bolivia, and no multilocus sequence typing (MLST) of pneumococcal isolates from this region has previously been documented. A pneumococcal carriage rate of 34% was identified. Of 53 Streptococcus pneumoniae isolates that survived transportation for serotyping, antibiotic susceptibility testing and MLST, the commonest serotypes were 6A (9%), 34 (8%), 4 (6%), 9A (6%), 10A (6%), 19A (6%), 23F (6%) and 38 (6%); overall, 26 different serotypes were identified. Antibiotic susceptibility testing by Etest demonstrated high levels of susceptibility to penicillin (93%), erythromycin (98%), vancomycin (100%), chloramphenicol (100%), tetracycline (96%) and trimethoprim/sulfamethoxazole (co-trimoxazole) (85%). MLST identified that the majority (57%) of viable isolates belonged to previously unrecognised sequence types that are currently unique to Bolivia.

An unusual infection in an injecting drug user.

Injecting drug users are prone to atypical infections. We present a case of septic thrombophlebitis secondary to Fusobacterium gonidiaformans infection in a heroin user, which demonstrates the frequently unusual nature of pathogens and presentations in this group of patients.

A novel pneumococcus with a new association.

A case of severe invasive pneumococcal disease in a 68 year old female is described. She presented following a holiday in Turkey with an uncommon but well documented complication of Streptococcus pneumoniae bacteraemia; Austrian's triad of meningitis, pneumonia and endocarditis. She then progressed to develop an atypical variant of Guillain Barre syndrome, never previously documented in association with pneumococcal disease. The pneumococcus was identified as serotype 6A and genetic typing by multi-locus sequence typing showed it to be a unique genetic profile (ST4533). We hypothesise that ST4533 may have resulted from genetic re-assortment from streptococci which had colonised the patient in the United Kingdom and bacteria encountered in Turkey. The ability to associate uncommon genotypes with uncommon clinical presentations may improve understanding of the pathogenesis of this organism, and this highlights the need for international invasive pneumococcal disease surveillance.

Observations regarding historical accounts of pneumococcal diseases due to serotypes 1 and 3.

Surveillance of the serotypes causing invasive pneumococcal diseases in the UK has indicated increasing incidence of serotype 1- and serotype 3-related disease in recent years. The introduction of a pneumococcal conjugate vaccine to the paediatric vaccination schedule in 2006, which did not cover these serotypes, has been regarded as a contributing factor. Serotypes 1 and 3 were perhaps the most extensively studied pneumococcal serotypes in the early twentieth century when pneumococcal serotyping began. Such historical observations are pertinent to our understanding of contemporary disease manifestations for these serotypes as many parallels can be seen between their behaviour in the early twentieth century and the early twenty-first century. There are many relevant lessons to be learned from these pre-antibiotic era descriptions and the observations of our predecessors.

Investigation and control of a cluster of penicillin non-susceptible Streptococcus pneumoniae infections in a care home.

Two elderly residents of a care home were hospitalised with pneumonia over a period of one month. They had bacteraemia with penicillin non-susceptible Streptococcus pneumoniae (PNSP) and both died. All residents and staff of the care home were screened for PNSP using nasopharyngeal swabs, with one resident and one member of staff found to be asymptomatic carriers. Oral rifampicin was given to the carriers. All four strains were found to be serotype 14, and multilocus sequence typing (MLST) showed ST2652, not previously detected in Scotland. Review of care home residents showed that pneumococcal vaccination coverage was low (63%). This is similar to rates found in those aged > or =65 years in the general population and needs to be improved upon.

Automation of MLST using third-generation liquid-handling technology.

The molecular characterization of bacterial pathogens of clinical significance is increasingly important. Methods, such as multilocus sequence typing (MLST), allow bacterial strains to be characterized during case clusters, for antibiotic-resistant strains to be monitored, and for the impact of new vaccines to be assessed. Our laboratory performs MLST on Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus. We have developed high-throughput automated methods to allow MLST to be performed in a time scale useful in a clinical setting. Here we describe the automation of MLST on a third-generation liquid-handling robot.

Truncated xpt gene present in invasive Streptococcus pneumoniae may have implications for MLST schemes.

A serotype 1 disease-causing pneumococcus possessing a truncated xanthine phosphoribosyltransferase (xpt) housekeeping gene is described. The deletion is within the gene region used for multi-locus sequence typing (MLST) and may have occurred through genetic transformation or capsule switch between clones. The identification of this deletion in a clinical isolate therefore warrants highlighting due to potential errors that may ensue in isolate characterization and due to the fact that deletions may occur in other genes in this or other species characterized by MLST.

Genetic relatedness of antibiotic-resistant pneumococci isolated during case clusters.

Multilocus sequence typing of Streptococcus pneumoniae associated with two case clusters of disease is reported here for the first time. Isolates from the first cluster were serotype 19F, resistant to penicillin and erythromycin, and were characterized as ST 320. Isolates from the second cluster were serogroup 4, resistant to ciprofloxacin, and were characterized as ST 206. Therefore, the isolates from these clusters were antibiotic-resistant, of serotypes infrequently isolated, and of uncommon sequence types.

A single nucleotide polymorphism identification assay for the genotypic characterisation of Neisseria meningitidis using MALDI-TOF mass spectrometry.

The ability of matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF) to identify virulent clones of meningococci quickly and accurately is investigated. A single nucleotide polymorphism (SNP) within the fumC gene which differentiates between the hypervirulent ET-15 strain and other ET-37 complex strains is used to determine the usefulness of this method. In this study, MALDI-TOF proved to be a fast, effective alternative to traditional DNA sequencing for the identification of an individual nucleotide.

Comparison of commercial DNA extraction kits for extraction of bacterial genomic DNA from whole-blood samples.

The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.

Nucleotide sequence analysis of the sialyltransferase genes of meningococcal serogroups B, C, Y and W135.

A rapid method for serogrouping meningococci is essential for the characterization of phenotypically non-groupable meningococcal isolates and clinical samples, particularly for public health management purposes. The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides serogrouping results of meningococcal isolates and clinical samples using a PCR assay which detects restriction fragment length polymorphisms in meningococcal serogroups B, C, Y and W135. Although this PCR system was invaluable when first introduced, it has several drawbacks and lacks the required sensitivity for detecting DNA in clinical samples. Due to the recent introduction of the meningococcal group C conjugate vaccine and an impending group B vaccine, a more robust and informative method for serogroup determination is required. A protocol was devised allowing PCR amplification of the siaD gene of serogroup B, C, Y and W135 meningococci. This system was multiplexed and allowed serogroup differentiation between serogroups B and C and also between B/C and Y/W135 by product size analysis. A nested stage was incorporated into the system for enhanced detection of meningococci in clinical samples, and finally a sequencing protocol was designed allowing detection of any nucleotide changes within the siaD gene. This system allows rapid serogrouping results for use within an agarose gel system as well as more informative results when used for sequencing within the siaD gene.

Analysis of PorA variable region 3 in meningococci: implications for vaccine policy?

Outer membrane protein (OMP) vaccines are being developed against Neisseria meningitidis serogroup B which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited data is available in Europe from genosubtyping meningococci. We therefore undertook a retrospective analysis of the three main variable regions, VR1, VR2 as well as VR3, of the porA gene from N. meningitidis isolated from different countries, mainly from Scotland and Sweden. Analysis of this gene showed that, amongst 226 strains studied, there were a total of 78 different strains. No new VR1 or VR2 alleles were found but five new VR3 alleles are described. Our data indicates the importance of analysing the VR3 region of PorA in addition to VR1 and VR2 and also highlights, in general terms, the need for genosubtyping meningococci. Such analyses have major implications for the design of new meningococcal vaccines.

Evaluation of a fluorescence-based PCR method for identification of serogroup a meningococci.

Standard and fluorescence-based PCR assays were developed for the identification of serogroup A meningococci by detection of the mynA gene. This assay was evaluated using bacterial cultures but provides the sensitivity required for the detection of the mynA gene from bodily fluids during meningococcal disease.

Detection and genotyping of meningococci using a nested PCR approach.

An effective vaccine against Neisseria meningitidis serogroup B is required. Outer-membrane protein vaccines have been developed, which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited genosubtyping data are available because most laboratories use mAbs directed against a limited number of specific serotypes and serosubtypes and laboratories do not genosubtype directly from body fluids due to the lack of a sensitive PCR method. A nested PCR was therefore developed that enables the amplification of the porA gene directly from clinical samples and has the required sensitivity for nucleotide sequencing of the three main variable regions, VR1, VR2 and VR3. Data were compared with those from culture-based nucleotide sequencing, and the use of this method increased the availability of genosubtype information by 45 %, thereby indicating the impact that this methodology has on the data provided and the implications for vaccine design.

Rapid assignment of nucleotide sequence data to allele types for multi-locus sequence analysis (MLSA) of bacteria using an adapted database and modified alignment program.

A novel database and modified alignment program is described which provides a fast and accurate procedure for assigning nucleotide sequences to allele types for multi-locus sequence analysis (MLSA). The database has between 40 and 160 alleles per organism including Neisseria meningitidis, Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae. The database directly compares the query nucleotide sequence against all alleles within the database and this system reduces the time taken for the analysis of nucleotide sequence data and assignment of alleles for subsequent sequence analysis.

Semi-automation of the polymerase chain reaction for laboratory confirmation of meningococcal disease.

Demand for accurate high-throughput detection and characterisation of medically important bacteria has increased dramatically within research and clinical laboratories. Liquid-handling robots have been developed to achieve high levels of accuracy and reproducibility. Assay automation can play a key role in the modern diagnostic laboratory and the data presented here shows that automated PCR is comparable with manual methods. Importantly, automation is preferred when high-quality results cannot be guaranteed using manual methods. This is particularly important when results are required quickly for public health management.