Ruptured ovarian ectopic pregnancy presenting with an acute abdomen.

An ectopic pregnancy occurs in 2% of all pregnancies. A primary ovarian ectopic (OP) is a rare entity and occurs in <2% of all ectopic gestations. It may present in those individuals who take ovulatory drugs, use an intrauterine device or have undergone in vitro fertilisation or embryo transfer. Multiparity and a younger age are other recognised risk factors. Diagnosing an OP pregnancy remains a challenge and it may be misdiagnosed as a bleeding luteal cyst, a haemorrhagic ovarian cyst or a tubal pregnancy by ultrasound scan. The diagnosis is often only established at laparoscopy following histopathological examination. A ruptured OP is a potentially life-threatening condition due to its potential for haemorrhage and hemodynamic collapse. Hence, early diagnosis is crucial to prevent serious morbidity and mortality. The authors present the case of a multiparous woman in her late 30s presenting with a seizure and lower abdominal pain at 6 weeks gestation. Her beta human chorionic gonadotropin was >9000 Miu/mL. A transvaginal ultrasound scan showed no evidence of an intrauterine pregnancy. There was free fluid in the pelvis. She was hemodynamically stable. She underwent a diagnostic laparoscopy, which showed hemoperitoneum and a ruptured left OP pregnancy. She underwent a left oophorectomy. Histology confirmed chorionic villi within the ovarian stroma. This case demonstrates the challenges in preoperative diagnosis of a ruptured OP pregnancy and acts as a cautionary reminder that individuals can present with hemodynamic stability. Rarely, as in this case, an OP pregnancy can occur without the presence of risk factors. Despite its rarity, a ruptured OP pregnancy should be considered in the differential diagnosis of women of reproductive age presenting to the emergency department with acute abdominal pain and a positive pregnancy test.

Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors.

Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.

A Role for Tetracycline Selection in Recent Evolution of Agriculture-Associated Clostridium difficile PCR Ribotype 078.

The increasing clinical importance of human infections (frequently severe) caused by Clostridium difficile PCR ribotype 078 (RT078) was first reported in 2008. The severity of symptoms (mortality of ≤30%) and the higher proportion of infections among community and younger patients raised concerns. Farm animals, especially pigs, have been identified as RT078 reservoirs. We aimed to understand the recent changes in RT078 epidemiology by investigating a possible role for antimicrobial selection in its recent evolutionary history. Phylogenetic analysis of international RT078 genomes (isolates from 2006 to 2014, n = 400), using time-scaled, recombination-corrected, maximum likelihood phylogenies, revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired a different allele of the tetracycline resistance gene tetM Consequently, an unusually high proportion (76.5%) of RT078 genomes were tetM positive. Multiple additional tetracycline resistance determinants were also identified (including efflux pump tet40), frequently sharing a high level of nucleotide sequence identity (up to 100%) with sequences found in the pig pathogen Streptococcus suis and in other zoonotic pathogens such as Campylobacter jejuni and Campylobacter coli Each RT078 tetM clonal expansion lacked geographic structure, indicating rapid, recent international spread. Resistance determinants for C. difficile infection-triggering antimicrobials, including fluoroquinolones and clindamycin, were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid, international spread via the food chain, may explain the increased RT078 prevalence in humans. Our work indicates that the use of antimicrobials outside the health care environment has selected for resistant organisms, and in the case of RT078, has contributed to the emergence of a human pathogen.IMPORTANCEClostridium difficile PCR ribotype 078 (RT078) has multiple reservoirs; many are agricultural. Since 2005, this genotype has been increasingly associated with human infections in both clinical settings and the community. Investigations of RT078 whole-genome sequences revealed that tetracycline resistance had been acquired on multiple independent occasions. Phylogenetic analysis revealed a rapid, recent increase in numbers of closely related tetracycline-resistant RT078 (clonal expansions), suggesting that tetracycline selection has strongly influenced its recent evolutionary history. We demonstrate recent international spread of emergent, tetracycline-resistant RT078. A similar tetracycline-positive clonal expansion was also identified in unrelated nontoxigenic C. difficile, suggesting that this process may be widespread and may be independent of disease-causing ability. Resistance to typical C. difficile infection-associated antimicrobials (e.g., fluoroquinolones, clindamycin) occurred only sporadically within RT078. Selective pressure from tetracycline appears to be a key factor in the emergence of this human pathogen and the rapid international dissemination that followed, plausibly via the food chain.

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection.

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.

Emergence and global spread of epidemic healthcare-associated Clostridium difficile.

Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance-conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

Is the colposcopically directed punch biopsy a reliable diagnostic test in women with minor cytological lesions?

OBJECTIVE: The study aimed to determine the accuracy of the colposcopy-directed punch biopsy (punch) to detect or exclude high-grade cervical intraepithelial neoplasia (CIN 2 or 3) in women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) cytological result and minor colposcopic findings. MATERIALS AND METHODS: In a diagnostic test accuracy study, women with ASCUS or LSIL cytological result and minor colposcopic changes had a single colposcopy-targeted punch biopsy was performed immediately followed by a loop electrocautery excision procedure (LEEP) biopsy. The trial was powered to detect a level of κ for a dichotomous outcome of 0.4 (i.e., fair-to-moderate agreement), with a two-sided significance level of 5% and a power of 90%. Accuracy parameters were computed using a cutoff for positive punch biopsy result of CIN 1+ and CIN 2+ for an outcome of CIN 2+ and CIN 3+ assessed in the LEEP specimen. RESULTS: Sixty-eight punch biopsy/LEEP-paired samples were analyzed. Of the 8 CIN 3 lesions, 6 and 4 were detected at cutoff CIN 1+ and CIN 2+, respectively (sensitivity, 50% and 75%). The corresponding specificities were 65% (39/60) and 97% (58/60). The punch biopsies identified only 14 (67%) or 6 (20%) of the 21 CIN 2+ lesions at cutoff CIN 1+ or CIN 2+, respectively. Of the punch biopsies, 31 (45.6%) accurately detected the severity of cervical abnormality. CONCLUSIONS: A single colposcopically directed punch biopsy appears to be insufficient to exclude underlying CIN 2 or 3.

Novel mucosal vaccines generated by genetic conjugation of heterologous proteins to pneumolysin (PLY) from Streptococcus pneumoniae.

Induction of immunity at mucosal surfaces is thought to be an essential feature in the protection of the host against the many pathogens that gain access through these surfaces. Here we describe how strong local and systemic immune responses can be generated when proteins are genetically conjugated to pneumolysin (PLY) from Streptococcus pneumoniae. Using green fluorescent protein (eGFP) and PsaA from S. pneumoniae, we have shown that genetic fusion (eGFPPLY and PsaAPLY) is essential to ensure high levels of antigen specific IgG and IgA in the serum and at mucosal surfaces. This form of vaccination is highly effective with antigen specific antibodies detected after a single dose of nanogram quantities of the conjugated proteins. In addition, generation of a non-toxic variant (eGFPDelta6PLY) indicated that while the toxic activity of PLY was not essential for adjuvanticity, it contributed to the magnitude of the response generated. Whilst vaccination with the PsaAPLY fusion proteins did not protect the animals from challenge, these studies confirm the utility of pneumolysin to act as a novel mucosal adjuvant to substantially increase the local and systemic humoral response to genetically fused protein antigens.

Synchrotron-based FTIR spectra of stained single cells. Towards a clinical application in pathology.

Over the last few years, FTIR spectroscopy has become a potential analytical method in tissue and cell studies for cancer diagnosis. This has opened a way towards clinical applications such as a tool that would scan samples to assess the presence or absence of malignant cells in biopsies, or as an aid to help pathologists to better characterise those cells that are suspicious but not diagnostic for cancer. The latter application has the problem that in order to assess these cells pathologists would have already dealt with stained samples. Therefore, it is important to understand how staining would affect the spectra of cells. To this purpose, we have conducted this study in order to clarify, first, how haematoxylin and eosin (H&E) and Papanicolau (Pap) stainings affect the spectra of single cells and, second, whether FTIR spectroscopy could differentiate between stained lung cancer cells and their normal counterparts. Furthermore, different cell preparations (cytospin, and smear) used in cytological diagnosis were assessed. Experiments performed using a bright infrared (IR) source (synchrotron) showed that both H&E and Pap staining induced marked changes in the lipid and amide-II band regions. Despite this, FTIR spectroscopy of already stained cells is capable of differentiating between lung cancer cells and their normal counterparts. The clinical applications of this methodology are discussed.

Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1.

Currently, the Golden Syrian hamster is widely considered an important model of Clostridium difficile disease, as oral infection of this animal pretreated with antibiotics reproduces many of the symptoms observed in humans. Two C. difficile strains, B1 and 630, showed significant differences in the progression and severity of disease in this model. B1-infected hamsters exhibited more severe pathology and a shorter time to death than hamsters infected with 630. Histological changes in the gut did not correlate with absolute numbers of C. difficile bacteria, but there were clear differences in the distribution of bacteria within gut tissues. Light, scanning, and transmission electron microscopy revealed high numbers of B1 bacteria at the mucosal surface of the tissue, whereas 630 bacteria were more frequently associated with the crypt regions. Both B1 and 630 bacteria were frequently observed within polymorphonuclear leukocytes, although, interestingly, a space frequently separated B1 bacteria from the phagosome wall, a phenomenon not observed with 630. However, pilus-like structures were detected on 630 located in the crypts of the gut tissue. Furthermore, B1 bacteria, but not 630 bacteria, were found within nonphagocytic cells, including enterocytes and muscle cells.

Multidisciplinary colposcopy clinicopathology correlation meetings: an activity review.

OBJECTIVE: Multidisciplinary colposcopy clinicopathology correlation meetings are deemed to be an important aspect of colposcopic quality assurance and are often a focus of attention in colposcopy quality assurance peer-review assessments. Despite this, there are few data on such meetings detailing activity or providing benchmarks for audit. MATERIALS AND METHODS: A retrospective analysis of clinicopathology correlation meetings held during a 3-year period (2004-2006) at the University Hospital of North Staffordshire was performed. RESULTS: A total of 65 meetings were held on a 2 to 4 weekly basis. All meetings contained a representation from cytology, pathology, and colposcopy. A total of 518 cases were listed and 475 were discussed, representing 6.6% of the total patient attendances at the colposcopy clinic during the study period. The main indications for discussion were as follows: cytology/histology discrepancy (35%), cytology/colposcopy discrepancy (10%), management dilemma (25%), and invasive cancer review (18%). A small proportion of cases listed (8%) were not discussed because of administrative problems. Problems were encountered in the quality of documentation, inconsistencies in the recording of findings, conclusions, and management plans. CONCLUSIONS: Multidisciplinary colposcopy pathology meetings provide a valuable data resource for recording and analyzing challenging areas in the clinical management of women with abnormal cervical cytology. However, such meetings are time and labor intensive both in terms of personnel and preparation. National guidelines need to be developed to guide clinicians on the frequency and standards required from such meetings.

A proteomic analysis of arsenical drug resistance in Trypanosoma brucei.

We have undertaken 2-DE and MS to identify proteins associated with arsenical drug resistance in Trypanosoma brucei. This parasite causes sleeping sickness in humans, and arsenical drug resistance is a significant potential problem. Comparative analysis of approximately 2000 spots resolved by 2-DE in the soluble proteomes of drug-sensitive and drug-resistant isogenic lines of T. brucei identified a protein spot whose absence associated with resistance to the arsenical drug, Cymelarsan. MS matched this protein to an identical pair of tandem genes Tb09.211.0120 and 0130 that encode a putative nascent polypeptide associated complex subunit. This protein also occurs as an isoform located in both resistant and sensitive lines at a similar molecular weight, but different pI. The difference between isogenic lines was confirmed by Western blot using an antibody against recombinant protein. Both genes were identical in sequence between drug-sensitive and drug-resistant lines and both were transcribed as determined by RT-PCR. We postulate that the missing protein isoform arose due to the lack of a PTM.

Construction and immunological characterization of a novel nontoxic protective pneumolysin mutant for use in future pneumococcal vaccines.

Pneumolysin, the pore-forming toxin produced by Streptococcus pneumoniae, may have an application as an immunogenic carrier protein in future pneumococcal conjugate vaccines. Most of the 90 S. pneumoniae serotypes identified produce pneumolysin; therefore, this protein may confer non-serotype-specific protection against pneumococcal infections such as pneumonia, meningitis, and otitis media. However, as pneumolysin is highly toxic, a nontoxic form of pneumolysin would be a more desirable starting point in terms of vaccine production. Previous pneumolysin mutants have reduced activity but retain residual toxicity. We have found a single amino acid deletion that blocks pore formation, resulting in a form of pneumolysin that is unable to form large oligomeric ring structures. This mutant is nontoxic at concentrations greater than 1,000 times that of the native toxin. We have demonstrated that this mutant is as immunogenic as native pneumolysin without the associated effects such as production of the inflammatory mediators interleukin-6 and cytokine-induced neutrophil chemoattractant KC, damage to lung integrity, and hypothermia in mice. Vaccination with this mutant protects mice from challenge with S. pneumoniae. Incorporation of this mutant pneumolysin into current pneumococcal vaccines may increase their efficacy.