Pathologic findings and causes of death in bottlenose dolphins Tursiops truncatus stranded along the Georgia coast, USA (2007-2013).

Between 2007 and 2013, before the 2013 cetacean morbillivirus outbreak, 26 fresh bottlenose dolphin carcasses were necropsied on the coast of Georgia, USA. Here, we present the pathological and microbiological findings associated with their most likely causes of death. The primary cause of death was determined in 25 individuals and included systemic bacterial infection (n = 7), verminous and bacterial bronchopneumonia (n = 5), drowning/entanglement (n = 5), disseminated histoplasmosis (n = 1), intestinal intussusception (n = 1), vegetative endocarditis (n = 1), meningitis (n = 1), necrotizing dermatitis (n = 1), disseminated angiomatosis (n = 1), emaciation (n = 1) and stingray spine trauma (n = 1). Histiocytic and eosinophilic bronchopneumonia associated with Halocerchus sp. infection was observed in 69% of the animals (18/26) and eosinophilic gastritis due to Anisakidae nematodes was found in 36% of the examined stomachs (8/22). Moderate to severe eosinophilic pancreatitis with fibrosis was observed in 4 animals infected with Brachycladiidae trematodes. Proliferative and ulcerative lymphoplasmacytic dermatitis was found in 5 animals and was considered to contribute to deteriorated health status in 2 calves. Pulmonary and lymph node angiomatosis were observed in 15 and 10 animals, respectively. In at least 2 animals, the concentration of polychlorinated biphenyls (PCBs) in the blubber exceeded 1500 µg g-1 of lipid. Bottlenose dolphins stranded on the Georgia coast have a wide range of inflammatory lesions associated with a variety of helminth, bacterial, and fungal pathogens. Some resident animals have also been exposed to high levels of PCB contamination, which could reduce host immunocompetence. Higher exposure to these or other pathogens could result in further decline in the health of resident and migrant dolphin populations in this region.

Antimicrobial susceptibility testing of historical and recent clinical isolates of Bordetella pertussis in the United Kingdom using the Etest method.

Reports of the development of antimicrobial resistance by Bordetella pertussis to macrolides in the United States and Taiwan, together with a recent increase in pertussis notifications and laboratory-confirmed cases in England and Wales in 2008, prompted the examination of historical and recent clinical isolates from patients for evidence of such resistance in our collection. Isolates submitted to our laboratory as part of the enhanced surveillance scheme for pertussis, from 2001 to 2009, were tested against three agents, erythromycin, clarithromycin and azithromycin, by the Etest (bioMérieux) method. All isolates (n = 583) were fully susceptible to all three agents tested (minimum inhibitory concentrations [MICs] 256 microg/ml. Although no evidence of resistance was found in the strains tested from the United Kingdom, screening for antimicrobial resistance of B. pertussis may be warranted in cases that are unresponsive to macrolide treatment and to provide early warning of such emergence in the future.

Pneumococcal serotypes causing pediatric meningitis in Turkey: application of a new technology in the investigation of cases negative by conventional culture.

The surveillance of serotypes causing invasive pneumococcal disease (IPD) provides further insight into the pathogenesis of pneumococcal disease and is important in order to track vaccine impact. Although the Quellung reaction has been accepted as the standard method for serotyping, prior antibiotic use causes a gap in studies based on bacterial culture. A total of 31 cerebrospinal fluid (CSF) samples found to be positive for Streptococcus pneumoniae by polymerase chain reaction (PCR) targeting the ply gene during an active surveillance were tested in a Bio-Plex multiplex antigen detection assay capable of detecting 14 serotypes/groups (1, 3, 4, 5, 6A, 6B, 7F/A, 8, 9V, 14, 18, 19A, 19F, and 23F). Twenty-seven CSF samples could be serotyped. The most common serotypes were serotypes 5 (n = 7), 19F (n = 5), 1 (n = 3), and 23F (n = 3). Theoretical coverage rates by the heptavalent (PCV7), 10-valent (PCV10), and 13-valent (PCV13) pneumococcal conjugate vaccines for bacterial meningitis were 48.1, 85.2, and 92.3%, respectively, for all age groups and 71.4, 85.7, and 100.0%, respectively, for those under 2 years of age. We propose that antigen detection assay used in conjunction with a PCR assay can be effectively applied in CSF samples to detect the pneumococcal serotypes, especially when the patient may have already been treated and, therefore, the cultures would be negative.

Genetic characteristics of pneumococcal disease in elderly patients before introducing the pneumococcal conjugate vaccine.

Streptococcus pneumoniae strains causing invasive pneumococcal disease (IPD) in the elderly population of England and Wales during the winter of 2003/2004 (1 November 2003 to 30 April 2004) were characterized by serotyping and genotyping in order to determine their population structure in the elderly. Serotyping and multilocus sequence typing (MLST) were carried out on 542 invasive isolates referred to the Respiratory and Systemic Infection Laboratory. Pneumococci were distributed among 32 serotypes and 144 MLST sequence types. A high genetic diversity was observed within the major serotypes. Genetic relatedness varied with regard to serotype. Isolates within serotypes 3, 7F and 8 were the most genetically related whereas serotypes 6A and 19F comprised isolates originating from unrelated ancestors. There was indirect evidence that some pneumococci were derived from clones that had undergone capsular switching in the past. Interestingly one case of IPD was caused by a pneumococcus originating from a clone that had undergone capsular switching from serotype 18C, a serotype included in 7-valent pneumococcal conjugate vaccine (PCV) to serotype 1 (serotype not included in PCV) suggesting that virulent clones with the potential ability to evade PCV existed in the pneumococcal population prior to the routine introduction of this vaccine. Isolates from 28 cases of apparent 23-valent pneumococcal polysaccharide vaccine (PPV) failure were included but there was no evidence of the emergence of particular clones associated with vaccine failures. Longitudinal studies based on serotypic and genetic characterization of pneumococci are fundamental to understanding the impact of both PPV and PCV on the genetic structure of pneumococcal populations.

The clinical and public health value of non-culture methods in the investigation of a cluster of unexplained pneumonia cases.

During 2003, a cluster of initially unexplained pneumonia cases (two fatal) occurred in patients aged <50 years in a British city. Routine culture tests were inconclusive, however, pneumococcal infection was suspected and the putative outbreak was investigated using non-culture methods. Clinical samples from ten patients were tested by pneumococcal polymerase chain reaction (PCR) and multi-locus sequence typing (MLST), or Binax NOW pneumococcal urine antigen test and serotype-specific enzyme-linked immunosorbent assay (ELISA). Lung samples from the deceased patients were PCR positive and yielded different MLST types. Two patients in one family group were serotype 1 pneumococcal antigen positive. Two further patients were serotype 1 antigen positive, and one serotype 4 positive. Two antigen-positive cases were also serum PCR positive. Non-culture methods confirmed the disease aetiology in six cases. Serotype and MLST results showed no single outbreak, but a family cluster of cases in a high background of pneumococcal pneumonia, providing important epidemiological data that would not otherwise have been available.

Trends in incidence of pneumococcal disease before introduction of conjugate vaccine: South West England, 1996-2005.

Introduction of pneumococcal conjugate and polysaccharide vaccines into the United Kingdom's routine immunization programmes is expected to change the epidemiology of invasive pneumococcal disease (IPD). We have documented the epidemiology of IPD in an English region (South West) with high-quality surveillance data before these programmes were established. We analysed data on isolates of Streptococcus pneumoniae from blood and CSF between 1996 and 2005 from microbiology laboratories in the South West that were reported and/or referred for serotyping to the Health Protection Agency Centre for Infections. The mean annual incidence of IPD increased from 11.2/100 000 in 1996 to 13.6/100 000 in 2005 (P<0.04). After adjusting for annual blood-culture sampling rates in hospitals serving the same catchment populations, an increase in annual incidence of IPD was no longer observed (P=1.0). Variation in overall incidence between laboratories could also be explained by variation in blood culture rates. The proportion of disease caused by serotypes 6B, 9V and 14 decreased significantly (P=0.001, P=0.007, and P=0.027 respectively) whereas that caused by serotype 4, 7F and 1 increased (P=0.001, P=0.003, and P<0.001 respectively) between 2000 and 2005. The level of penicillin non-susceptibility and resistance to erythromycin remained stable (2% and 12% respectively). This study provides an important baseline to assess the impact of changing vaccination programmes on the epidemiology of IPD, thus informing future use of pneumococcal vaccines.

The contribution of respiratory pathogens to the seasonality of NHS Direct calls.

OBJECTIVES: Primary care is thought to bear half the cost of treating infections in the UK. We describe the seasonal variation in NHS Direct respiratory calls (a new source of primary care data) and estimate the contribution of specific respiratory pathogens to this variation. METHODS: Linear regression models were used to estimate the weekly contribution of specific respiratory pathogens to the volume of NHS Direct respiratory calls (England and Wales, 2002-2004, all ages and 0-4 years). RESULTS: Annual peaks in NHS Direct cough and difficulty breathing calls occurred in late December, with peaks in 'cold/flu' and fever calls occurring between November and April. The main explanatory variables were influenza (estimated to account for 72.5 calls per 100,000/year; 22% of 'cold/flu' calls; 15% of cough; and 13% of fever) and Streptococcus pneumoniae (55.5 per 100,000; 33% of 'cold/flu' calls; 20% of cough; and 15% of fever (0-4 years)). CONCLUSIONS: It is estimated that respiratory viruses, notably influenza and RSV, are responsible for at least 50% of the seasonal variation in NHS Direct respiratory calls. These results provide estimates of the burden of specific respiratory diseases reported to NHS Direct, and will help interpret syndromic surveillance data used to provide early warning of rises in community morbidity.

Invasive group A streptococcal disease: should close contacts routinely receive antibiotic prophylaxis?

Group A streptococci (Streptococcus pyogenes) causes a wide range of illnesses from non-invasive disease--eg, pharyngitis--to more severe invasive infections--eg, necrotising fasciitis and toxic shock-like syndrome. There remains uncertainty about the risk of secondary cases of invasive disease occurring among close contacts of an index case and how best to manage that risk. We do not consider that currently available evidence justifies the routine administration of chemoprophylaxis to close contacts. We suggest that the appropriate response should be to routinely inform all household contacts of a patient with invasive group A streptococcal disease about the clinical manifestations of invasive disease and to seek immediate medical attention if they develop such symptoms.

Antipsychotic drug treatment alters expression of mRNAs encoding lipid metabolism-related proteins.

Using an automated PCR-based genomics approach, TOtal Gene expression Analysis (TOGA), we have examined gene expression profiles of mouse striatum and frontal cortex in response to clozapine and haloperidol drug treatment. Of 17 315 mRNAs observed, TOGA identified several groups of related molecules that were regulated by drug treatment. The expression of some genes encoding proteins involved in neurotransmission, signal transduction, oxidative stress, cell adhesion, apoptosis and proteolysis were altered in the brains of both clozapine- and haloperidol-treated mice as recognized by TOGA. Most notable was the differential expression of those genes whose products are associated with lipid metabolism. These include apolipoprotein D (apoD), the mouse homolog of oxysterol-binding protein-like protein 8 (OSBPL8), a diacylglycerol receptor (n-chimerin), and lysophosphatidic acid (LPA) acyltransferase. Real-time PCR analysis confirmed increases in the RNA expression of apoD (1.6-2.2-fold) and OSBPL8 (1.7-2.6-fold), and decreases in the RNA expression of n-chimerin (1.5-2.2-fold) and LPA acyltransferase (1.5-fold) in response to haloperidol and/or clozapine treatment. Additional molecules related to calcium homeostasis and signal transduction, as well as four sequences of previously unidentified mRNAs, were also confirmed by real-time PCR to be regulated by drug treatment. While antipsychotic drugs may affect several metabolic pathways, lipid metabolism/signaling pathways may be of particular importance in the mechanisms of antipsychotic drug action and in the pathophysiology of psychiatric disorders.

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK.

OBJECTIVE: To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK. METHODS: During 1995-97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone. RESULTS: Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mefA gene was detected in seven isolates (five serotype 14 and two serotype 19F), ermB in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone. CONCLUSIONS: The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.

Emergence of a fluoroquinolone-resistant strain of Streptococcus pneumoniae in England.

OBJECTIVE: To determine the epidemiological relationship between pneumococci of serotype 9V, with reduced susceptibility to ciprofloxacin, penicillin and erythromycin, referred to the Reference Laboratory during 1997-2001. METHODS: Isolates were characterized by multilocus sequence typing (MLST), PFGE, and sequencing of parC and gyrA. Relevant clinical data were sought. RESULTS: Forty-eight isolates were received from nine laboratories in England, but 35 (73%) were from one laboratory in Birmingham, and were mostly from elderly patients receiving ofloxacin or ciprofloxacin for respiratory infections. There were two quinolone resistance phenotypes, with ciprofloxacin, moxifloxacin and gemifloxacin MICs of 8-32, 0.5-1 and 0.125-0.25 mg/L, and 64-256, 4-16 and 1-4 mg/L, respectively. Each of three isolates from the former group had mutations in parC, whereas each of nine isolates from the more resistant group had mutations in both parC and gyrA. Several also had increased quinolone efflux. Typing of 27 quinolone-resistant isolates showed that eight were indistinguishable from the epidemic Spain9V-3 (ST156) clone, while the remainder belonged to a novel but related type (ST609), that differed from Spain9V-3 at 2/7 alleles (2 bp changes in aroE and 1 bp change in gdh). Both MLST types were represented among isolates with high- and low-level quinolone resistance. Three of five serotype 9V isolates from Birmingham, with reduced susceptibility to penicillin and erythromycin, and ciprofloxacin MICs of 1-2 mg/L, belonged to MLST type ST609, while another was indistinguishable from the Spain9V-3 clone. Review of records of 32 patients from Birmingham indicated that some isolates were nosocomial, whereas others were acquired in the community. CONCLUSIONS: In the late 1990s, a quinolone-resistant strain, clonally related to Spain9V-3, emerged in England, principally in Birmingham.

Diagnosis of invasive pneumococcal infection by PCR amplification of Streptococcus pneumoniae genomic fragments in blood: a multi-centre comparative study.

The increasing interest in the prevention of pneumococcal disease by immunisation necessitates improved organism-specific surveillance. This is particularly the case with regard to the contribution of Streptococcus pneumoniae infection to community-acquired pneumonia where blood cultures are often negative and sputum culture results ambiguous. Examination by PCR of blood samples taken at hospital admission offers one possibility for such improvement. The sensitivity, specificity and convenience of three pneumolysin gene PCR assays were compared in a large study, using EDTA blood from 175 patients (95 with proven pneumococcal bacteraemia, 80 with bacteraemia due to other organisms). The assays used were a PCR-enzyme immunoassay, a hybridisation probe assay run on the Roche LightCycler and a hydrolysis probe (TaqMan) assay run on an ABI 7700. Overall samples from only 57% of patients with bacteraemic pneumococcal infection yielded a positive result in at least one assay. Individual sensitivities ranged from 45% (TaqMan/ABI) through 35% (PCR-EIA) to 21% (Hybridisation/LightCycler). Specificity (PCR negative in the 80 control patients) ranged from 97-100%. The TaqMan/ABI assay was run in two centres and concordance between results was 91.4%, discrepancies being associated with very weakly positive samples. Overall, the TaqMan/ABI was the most sensitive and convenient assay; however, this method does not appear to offer any significant improvement over conventional blood cultures and is unlikely to be sufficiently sensitive to confirm a pneumococcal aetiology for non-bacteraemic pneumococcal pneumonia. For the present, therefore, blood culture is the preferred option.

An outbreak of serious illness and death among injecting drug users in England during 2000.

An outbreak of serious illness and death occurred in injecting drug users during 2000 in Scotland, Ireland and England. National and international collaboration was necessary for the investigation and management of this outbreak. In England and Wales active case-finding was initiated, coupled with standardised data collection and microbiological investigation of cases. Twenty-six definite or probable cases were identified in England between 1 April and 31 Aug. 2000; 17 of these occurred in the North. The overall case fatality was 50% (13/26). The principal apparent risk factor was a history of intramuscular or subcutaneous injection of heroin and the limited duration of the outbreak suggested that the problem might have been related to a particular supply of heroin. Clostridium novyi was isolated from two English cases. Taken in conjunction with contemporaneous microbiological and epidemiological results from Scottish and Irish cases, the probable aetiology for this outbreak was infection with C. novyi associated with both a particular supply of heroin and the method of preparation and injection used. A 'toolkit' was distributed in Sept. 2000 to all Consultants for Communicable Disease Control in England and Wales to assist them with the ongoing surveillance, investigation and management of this condition. Lessons learned have been used to produce guidance for the investigation and management of outbreaks of unexplained serious illness of possible infective aetiology.

Isolation and identification of Clostridium spp. from infections associated with the injection of drugs: experiences of a microbiological investigation team.

Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease. C. novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly. The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification. Additional phenotypic tests that are useful in recognising the main pathogenic species are described. Differentiation of C. novyi type A from C. botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture.

Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C. perfringens and Bacillus cereus isolated from injecting drug users during 2000.

As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

Correlations between metal uptake in the soft tissue of Perna perna and gill filament pathology after exposure to mercury.

The accumulation of metal in soft tissues, filtration rate and gill filament morphology are correlated in the southern African rock mussel, Perna perna, during exposure to mercury (24 days) and recovery (24 days). The amount of Hg in soft tissues increased from 0.13 to 87.5 microg/g dry weight after 24 days exposure, and declined to 13 microg/g during recovery. Mean filtration rate fell from 3,979 to 1,818 ml/h/g dry weight by day 2, but recovered slightly through days 4 and 8 (3,037 ml/h/g), with a higher average rate (5,030 ml/h/g) being maintained over the 24-48 days recovery period. The initial decline in filtration coincided with epithelial cell deterioration presented as interstitial oedema, neural and epithelial cell degeneration and reduced ciliation. Between days 8 and 24, cilia regenerated and there was a general improvement in cell morphology. Gill filament morphology returned to near normal during the metal-free recovery period. The usefulness of P. perna as an indicator of pollution is discussed.

Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria.

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.