Guidelines for the public health management of typhoid and paratyphoid in England: practice guidelines from the National Typhoid and Paratyphoid Reference Group.

OBJECTIVES: The Typhoid and Paratyphoid Reference Group (TPRG) was convened by the Health Protection Agency (HPA) and the Chartered Institute of Environmental Health (CIEH) to revise guidelines for public health management of enteric fever. This paper presents the new guidelines for England and their rationale. METHODS: Methods include literature reviews including grey literature such as audit data and case studies; analysis of enhanced surveillance data from England, Wales and Northern Ireland; review of clearance and screening schedules in use in other non-endemic areas; and expert consensus. RESULTS: The evidence and principles underpinning the new guidance are summarised. Significant changes from previous guidance include: • Algorithms to guide risk assessment and management, based on risk group and travel history; • Outline of investigation of non-travel cases; • Simplified microbiological clearance schedules for cases and contacts; • Targeted co-traveller screening and a "warn and inform" approach for contacts; • Management of convalescent and chronic carriers. CONCLUSIONS: The guidelines were launched in February 2012. Feedback has been positive: the guidelines are reported to be clear, systematic, practical and risk-based. An evaluation of the guidelines is outlined and will add to the evidence base. There is potential for simplification and consistency between international guidelines.

Imported chicken meat as a potential source of quinolone-resistant Escherichia coli producing extended-spectrum beta-lactamases in the UK.

OBJECTIVES: Escherichia coli producing CTX-M-15 enzyme began to rapidly spread in the UK from around 2003 but other types also occur, notably CTX-M-14. We examined breasts from UK-reared (n = 62) and imported (n = 27) chickens as potential sources of quinolone-resistant E. coli with bla(CTX-M) genes. A further 40 samples for which the country of rearing could not be identified were examined. METHODS: During 2006, 129 fresh and frozen chicken breast fillets were purchased from retail outlets in the West Midlands. These were cultured for E. coli on CLED agar containing 8 mg/L ciprofloxacin and carrying a 10 microg cefpodoxime disc. Resistant isolates were identified and typed by RAPD fingerprinting; bla(CTX-M) was identified by PCR and genotyped by reverse-line hybridization. RESULTS: The country of rearing was identified from the packaging for 89 of 129 purchased samples. Only one of the 62 UK-reared chicken samples carried E. coli producing a CTX-M-1 enzyme, whereas 10 of 27 samples reared overseas had E. coli with CTX-M enzymes. Specifically, 4/10 Brazilian, 3/4 Brazilian/Polish/French, and 2/2 Dutch samples had E. coli with CTX-M-2 enzymes. Six of 40 samples for which the country of rearing was not known had producers of CTX-M enzymes, 5 of them with CTX-M-14. CONCLUSIONS: Quinolone-resistant E. coli with various CTX-M beta-lactamase genes that are common in human infections worldwide were found in imported chicken breasts, indicating a possible source for gut colonization. Samples from Brazil were commonly positive for E. coli with CTX-M-2, the dominant bla(CTX-M) genotype from human infections in South America, which is currently rare in clinical infections in the UK. CTX-M-15, the dominant CTX-M type in human infections in the UK, was not found in chicken isolates, suggesting that the UK-reared chickens are not a reservoir of CTX-M-15.

TGF-beta inhibition of CTL re-stimulation requires accessory cells and induces peroxisome-proliferator-activated receptor-gamma (PPAR-gamma).

Effective cellular immunity to Epstein-Barr virus (EBV), necessary to prevent or cure many post-transplant lymphoproliferative disorders (PTLD), can be inhibited by transforming growth factor-beta (TGF-beta). In vitro, TGF-beta inhibits memory CTL re-stimulation from whole PBMC. We show that the effect of TGF-beta on CTL re-stimulation is not directly on the T cell, but requires an accessory cell (AC) population. Further, pre-treatment of AC with TGF-beta significantly reduces memory CTL re-stimulation and suppresses delayed type hypersensitivity (DTH) responses. Addition of exogenous interferon-gamma to the AC overcomes the effects of TGF-beta. TGF-beta pre-treatment also up-regulates expression of peroxisome-proliferator-activated receptor-gamma (PPAR-gamma) in CD14(+) AC. Importantly, pre-treatment of AC with the PPAR-gamma ligand, ciglitazone, results in significantly reduced memory CTL re-stimulation. Thus, the effects of TGF-beta in this system may be mediated in part via PPAR-gamma, and PPAR-gamma activation could have significant inhibitory effects on memory T-cell responses by affecting AC function. These data have important implications in understanding how memory CTL are re-stimulated and function to prevent disease, especially PTLD.

Provision and decontamination of uniforms in the NHS.

AIM: To ascertain the provision and decontamination of uniforms within a cross-section of NHS trusts in the UK and to compare policies regarding their use. METHOD: A questionnaire was circulated to 170 NHS trust infection control teams in the UK. Eighty-six (51 per cent) responses were received, which represented 101 NHS trusts. RESULTS: Less than half of the trusts (47 per cent) provide adequate numbers of uniforms to allow a clean uniform per shift. Only 26 per cent had adequate on-site staff changing facilities and 65 per cent did not launder uniforms. The majority of nursing staff (91 per cent) were compelled, by a combination of these factors, to launder their uniforms at home. Few were provided with any guidance on how to do this safely. CONCLUSION: There is an urgent need for minimum standards to be set for the provision of uniforms, laundering and changing facilities, to minimise the potential for spread of healthcare-associated infections.

Meningitis without a petechial rash in children in the Hib vaccine era.

AIMS: (1) To determine the causes of meningitis in children immunized with Hib vaccine, presenting without a non-blanching rash; (2) to review the use of dexamethasone in this group. METHOD: Retrospective review of all children with more then 10 white cells/mm(3) in their cerebrospinal fluid (CSF), admitted between January 1998 and August 2002. Children were excluded if they had a non-blanching rash on admission or if their discharge diagnosis was not meningitis. Local guidelines recommended dexamethasone to be given before antibiotics for children with meningitis and no rash. RESULTS: One hundred and eight children were identified. Causes of proven meningitis were: viral 41 (enterovirus 40), bacterial 22. CSF culture or PCR was the only diagnostic test in 31 children. Dexamethasone was given to 16 children. Length of admission was shorter in children with viral compared with bacterial meningitis (4 vs 8 days; P < 0.0001). SUMMARY: Viral meningitis is the commonest cause of meningitis without rash. Enteroviral PCR was the most useful test and needs to be widely available. Confirmation of enteroviral meningitis allowed early discharge. Few children were given dexamethasone, but only 5/108 may have benefited. CONCLUSIONS: The most common cause of meningitis without a rash in British children is enterovirus. The use of dexamethasone in children with meningitis without a rash should be reconsidered or, at least, individualised.

A comparison of the performance of commercially available chromogenic agars for the isolation and presumptive identification of organisms from urine.

AIMS: To compare four media-UTI medium, BBL CHROMagar, CPS ID2, and Harlequin CLED-using a collection of fully characterised organisms and subsequent "field trial". METHODS: Seven hundred and eighty seven fully characterised isolates (730 Gram negative bacteria, 47 Gram positive bacteria, and 10 yeasts) were used to test for accuracy of organism identification. To assess isolation rates and ability to detect mixed cultures, 1435 urine samples were cultured in the three best performing chromogenic media (UTI medium, BBL CHROMagar, and CPS ID2) and CLED. RESULTS: The chromogenic agars differed in their accuracy of identification, with BBL CHROMagar performing best and Harlequin CLED performing least well. Similarly, BBL CHROMagar achieved a higher overall isolation rate than UTI medium and CPS ID2. When mixed growth was defined as greater than two organism types, BBL CHROMagar detected more mixed cultures than did UTI medium and CPS ID2, although the differences were not significant. When mixed growth was defined as greater than one organism type the increased number of mixed growths detected by BBL CHROMagar became significant, largely because of differences in enterococcal isolation rates. CONCLUSION: The use of BBL CHROMagar, UTI medium, or CPS ID2 chromogenic agar as a replacement for CLED agar would improve the detection rate of contaminated urine samples. Enhanced identification helps to distinguish different species, facilitating the monitoring of bacterial resistance in support of the national antibiotic strategy. BBL CHROMagar gave the highest overall organism recovery rates, greatest ability to detect mixed cultures, and the most accurate identification of organisms.

Comparison of the performance of selenite cystine and mannitol selenite enrichment broths in the isolation of Salmonella spp. from faeces.

Over a four-month period, 4,658 routine faecal samples were examined in four laboratories and the isolation rates of Salmonella spp. from mannitol selenite (MS) and selenite cystine (SC) broths plated to xylose lysine desoxycholate agar (XLD) compared. The isolation rate by MS was 1.55% and by SC was 1.48%, a small difference which is not statistically significant. Significantly fewer colonies were selected for supplementary testing from SC than MS (p = 0.029), thus reducing confirmatory work. In laboratories where SC is already used for food and environmental work, an opportunity exists to limit stocked salmonella enrichment broths to SC alone.

A comparison of the performance of cystine lactose electrolyte deficient (CLED) agar with Oxoid chromogenic urinary tract infection (CUTI) medium for the isolation and presumptive identification of organisms from urine.

AIMS: As part of the UK antimicrobial resistance strategy and action plan, the Public Health Laboratory Service (PHLS) is required to collect antibiotic susceptibility data so that resistance trends and patterns can be monitored. Most laboratories report urine Gram negative isolates, as "coliforms" according to morphological appearance, but without an acceptable identification system the antimicrobial surveillance data will be meaningless. Commercially available identification systems tend to be expensive and time consuming. Chromogenic agars, which claim to improve the detection of mixed cultures and identification of organisms from urine, have now become available and may provide a cost effective alternative. The primary aim of this study was to compare the performance of cystine lactose electrolyte deficient (CLED) agar with a chromogenic agar (Oxoid urinary tract infection medium; CUTI) in terms of isolation rates and ability to detect mixed cultures. Secondary aims were to evaluate the correlation of "presumptive" identification of isolates from chromogenic media with that of two commercial identification systems and to appraise the sensitivity of the semiquantitative loop and filter paper strip culture techniques. METHOD: One thousand, four hundred and sixty six urine samples were examined in four laboratories using the semiquantitative culture methods of 1 microl loop and filter paper strip. The degree of accuracy of organism identification was measured by comparing the presumptive identification using colony colour supplemented with simple bench tests, with identification obtained from two more complex commercial systems. RESULTS: There was no significant difference between the performance of the loop and filter paper strip methods on the CLED agar, but the CUTI agar performed significantly better than the CLED agar for the detection of significant isolates and mixed cultures. This difference was greater using the loop method. Identification of the organisms using the commercial systems gave > 99% agreement and was therefore considered suitable as a standard against which to compare the presumptive CUTI identification. Using the manufacturer's colony colour criteria in combination with a bench indole test, the CUTI medium was 99% specific for Escherichia coli, although this was reduced to 97% if the indole test was omitted. Citrobacter spp were the most commonly misidentified organisms, giving false presumptive identification as E coli. By testing oxidase activity to differentiate Pseudomonas spp and the absence of indole production to support the identification of Proteus mirabilis, the CUTI medium provided a suitable identification for 86.8% of Gram negative isolates. The remaining 13.2% would require further identification. CONCLUSION: CUTI medium improves the detection of mixed cultures, thereby improving the reliability of reporting of significant isolates when compared with CLED agar. When supplemented with simple bench tests it provides an identification system capable of speciating 86.8% of Gram negative isolates and providing a valuable cost effective mechanism for antimicrobial resistance surveillance.

Methods for detecting apoptosis in thyroid diseases.

Over the last few years, the importance of apoptosis in determining the fate of thyrocytes in autoimmune thyroid disease has been the topic of intense investigation. It is now clear that thyrocytes from patients with Hashimoto's thyroiditis are destroyed as a result of an apoptotic process. However, there is no general consensus on whether the intrathyroidal lymphocytes or the thyrocytes themselves are responsible for their death. The use of a wide range of techniques has contributed to the assessment of this process both in situ on thyroid sections and in vitro on thyroid cell preparations. The apoptosis field of research is rapidly evolving and as the pathways to cell death become unravelled, novel methods will emerge. As each technique offers some advantage, it is critical to know the most suitable method for a specific study. Equally, each method also has intrinsic limitations. Thus, to achieve reliable results, it is necessary to use more than one technique per study. In addition, techniques related to the measurement of the expression of pro-apoptotic and anti-apoptotic genes have been contributing to the study of the susceptibility of the cells to apoptosis and/or to their ability to kill themselves or neighbouring cells. In this review we will focus on the most relevant techniques.

An evaluation of the performance of XLD, DCA, MLCB, and ABC agars as direct plating media for the isolation of Salmonella enterica from faeces.

AIMS: To compare the performance of four media, singly and in combination, as direct plating media for the isolation of Salmonella enterica from human faeces. METHODS: Two thousand four hundred and nine routine, faecal samples received by four laboratories were inoculated on to xylose lysine desoxycholate (XLD), desoxycholate citrate (DCA), mannitol lysine crystal violet brilliant green (MLCB), and alpha-beta chromogenic (ABC) agars using standardised protocols, reagents, and data collection. Isolates of presumptive salmonellae were identified using standard laboratory techniques and the results were analysed statistically. RESULTS: Direct plating recovered 46 of the 60 possible isolates of Salmonella spp recovered via enrichment broth. No isolates were recovered from direct plating that were not recovered via selenite enrichment. MLCB gave the highest isolation rate individually (84.8%) and amounts of competing flora (CF) did not affect the recognition of colonies. ABC proved highly specific, but insensitive, and isolation rates were adversely affected by any amount of CF. Isolation rates from XLD and DCA were only affected when the CF load was heavy. DCA was least specific, with only 9.01% of picks positive and greatest number of confirmatory tests. XLD and MLCB, in combination, gave the highest isolation rate. CONCLUSIONS: Where the earlier results of direct plating may be advantageous, XLD and MLCB provide the optimal combination. For non-typhi salmonellae, MLCB is the best, single direct plating medium. For routine diagnostic work, XLD is most effective.

Effect of incubation temperature on the isolation of thermophilic species of Campylobacter from faeces.

Although no large-scale clinical study has been performed, it has been reported that incubation at 37 degrees C gives better isolation rates for all common species of Campylobacter than incubation at 42 degrees C, while also improving the recovery of the more unusual species. In this study, 2,570 faecal samples were examined in four laboratories, using a standardised protocol. Isolation rates of Campylobacter spp. were compared after incubation on modified campylobacter blood-free selective agar at 37 degrees C and 42 degrees C. Campylobacter spp. isolates were made from 185 samples (7.2%); 25 were recovered only at 42 degrees C and three only at 37 degrees C (p < 0.001). There were significantly more colonies at 42 degrees C (p = 0.001). Competing flora were heavier at 37 degrees C, but this did not account for the difference in isolation rates or quantity of growth. It is recommended that cultures for Campylobacter spp. be routinely incubated at 42 degrees C. More specific techniques are required to seek for fastidious campylobacters.

Comparison of the performance of lactose and mannitol selenite enriched broths, subcultured to DCA and XLD agars, in the isolation of Salmonella spp. from faeces.

Selenite-based enrichment broths using either lactose or mannitol as a carbohydrate source are generally used as selective enrichment media for the isolation of Salmonella spp. from human faeces in the UK, but few studies have compared the relative efficacy of the available formulations. A variety of solid media is used for the routine subculture from these selective broths, but similarly we have been unable to find published evidence as to which medium performs best. Four thousand and nineteen faecal samples were examined in four laboratories and the isolation rates of Salmonella spp. from lactose (LS) or mannitol selenite (MS) broths, plated onto either xylose lysine desoxycholate agar (XLD) or desoxycholate citrate agar (DCA) were compared. MS performed significantly better than LS (p = 0.02), recovering 95 salmonellae compared with 87. No significant difference in isolation rates was found between XLD and DCA, although colonial appearances of suspected salmonellae on XLD were much more specific, resulting in significantly fewer colonies having to be selected for supplementary testing (p < 0.001) and so reducing confirmatory work. An opportunity exists to simplify holdings of media by choosing to use the MS/XLD combination.

Cryptosporidium meleagridis from humans: molecular analysis and description of affected patients.

OBJECTIVES: To genetically characterize an unusual genotype of Cryptosporidium from the stools of humans with diarrhoea and to identify risk factors in the affected patients. METHODS: DNA was extracted from human faeces where Cryptosporidium oocysts were detected by light microscopy. Cryptosporidial gene fragments from six different loci were analysed by PCR alone, PCR/RFLP and by DNA sequencing. Oocysts were characterized by light and immunofluorescence microscopy and epidemiological data was collected from the affected patients. RESULTS: Analysis of the Cryptosporidium oocyst wall protein (COWP) gene amplified from > 2000 human faecal samples identified 19 patients all of which produced an unusual RFLP profile. Subsequent DNA sequence analysis of this and an additional four genetic loci (including 18S rRNA sequences) confirmed these as a homogeneous group which was genetically distinct from Cryptosporidium parvum. The isolates were identified as Cryptosporidium meleagridis since the gene sequences were identical to those from this species recovered from birds. Conventional microscopy showed oocysts indistinguishable from C. parvum and reacted strongly with two different commercially available anti-oocyst monoclonal antibodies. None of the patients showed risk factors unusual for cryptosporidiosis; however, ten of the cases occurred during the summer/autumn, six had a history of foreign travel, four were co-infected with Giardia, two were HIV positive, and six were without identifiable immunocompromising factors. CONCLUSIONS: This study further confirms that C. meleagridis, in addition to C. parvum, is involved in human disease. The study also highlights the lack of basic information on the host range of this genus of parasites, the complexity of the transmission routes involved in human cryptosporidiosis, and the value of molecular techniques in identify hitherto unrecognised differences in Cryptosporidium from human faeces.

A comparison of the isolation rates of Salmonella and thermophilic Campylobacter species after direct inoculation of media with a dilute faecal suspension and undiluted faecal material.

Regardless of media used, dilution of faecal samples before direct plating may improve isolation rates and reduce subcultures by freeing organisms from the faecal mass and diminishing competing flora. Despite the routine use of dilution in many laboratories, it has never been established properly whether direct or dilute inocula should be used in primary plating of faeces. A total of 3764 faecal samples was examined in four laboratories with a standardised methodology. The isolation rates, competing flora and confirmatory work performed for Salmonella spp. and Campylobacter spp. from primary plating media with a dilute faecal inoculum were compared with those after direct inoculation of faecal material. Inoculum effects on the isolation of Shigella spp. could not be assessed as only one isolate occurred during the study period. The overall isolation rates of both major enteric pathogens were unaffected by the inoculum. However, significantly fewer wasted subcultures were recorded with a dilute inoculum for Campylobacter spp., and competing florawas reduced in all cases without diluting out small numbers of the pathogen.

A comparison of the performance of bacitracin-incorporated chocolate blood agar with chocolate blood agar plus a bacitracin disk in the isolation of Haemophilus influenzae from sputum.

The lack of selectivity of chocolated blood agar (CBA), routinely used for the isolation of Haemophilus influenzae, may lead to masking of the growth of H. influenzae due to overgrowth of competing flora. Bacitracin can be used as a selective agent, either incorporated into the medium or applied to the medium in a filter paper. However, neither method has been evaluated or compared in a large study. Sputum samples (1990) were examined in four laboratories and the isolation rates of H. influenzae on chocolated blood agar with bacitracin added to the medium (BCA) and chocolated blood agar (CBA) with a bacitracin disk were compared. A plain blood agar plate was also inoculated to facilitate the isolation of Streptococcus pneumoniae so that its effects on the isolation of H. influenzae could be assessed. No significant difference was found between the isolation rates of H. influenzae on BCA and CBA with a bacitracin disk, although competing flora was greatly reduced and quantity of growth of H. influenzae increased on BCA. The presence of S. pneumoniae did not affect the isolation of H. influenzae in this study.

Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. PHLS Advisory Committee for Food and Dairy Products.

These guidelines for the microbiological quality of ready-to-eat foods represent a revision and expansion of guidelines first published by the PHLS in September 1992 and revised in March 1996. The latest guidelines incorporate many of the constructive comments received from food examiners and other microbiologists within and outside the PHLS and from environmental health officers throughout the United Kingdom. This document reviews the changes and the reasons they were made and sets out the new guidelines. It also clarifies the role of food examiners in interpreting the microbiological results of formal samples.

A comparison of blood agar supplemented with NAD with plain blood agar and chocolated blood agar in the isolation of Streptococcus pneumoniae and Haemophilus influenzae from sputum. Bacterial Methods Evaluation Group.

Streptococcus pneumoniae grows well and generally exhibits typical morphology on Columbia blood agar, whereas Haemophilus influenzae requires a more complex medium to meet its growth requirements - usually chocolated blood agar - on which S. pneumoniae is less easily recognisable. Therefore, a single medium that produces typical morphology of S. pneumoniae and facilitates the growth of H. influenzae would have considerable potential advantages. It has been claimed that blood agar supplemented with nicotinamide adenine dinucleotide (NAD) is such a medium. However, despite its routine use in several large diagnostic laboratories its performance has never been properly evaluated. In the present study, 1724 sputum samples were examined in four laboratories. The isolation rates of H. influenzae and S. pneumoniae on NAD-supplemented blood agar (SBA) were compared with those on a two-plate combination of plain blood (BA) and chocolated blood agar (CBA). The two-plate combination performed significantly better for both organisms; isolation rates for H. influenzae were increased from 8.16% on SBA to 11.07% on BA plus CBA and for S. pneumoniae from 4.18% to 4.68%. Isolation rates were also compared after incubation for 24 and 48 h. With the two-plate combination, isolation rates for H. influenzae and S. pneumoniae were increased by 0.98% and 0.16%, respectively, and for SBA by 0.57% and 0.32% after 48 h. However, despite this increase, SBA still performed less well than the two-plate combination.

Human immunodeficiency virus-1 infection requires pertussis toxin sensitive G-protein-coupled signalling and mediates cAMP downregulation.

The human immunodeficiency virus-1 (HIV-1) utilises CD4 and certain beta-chemokine receptors, mainly CCR-5 and CXCR4, for attachment and virus entry into T-lymphocytes and monocytes/macrophages. CD4 and beta-chemokine receptors participate in intracellular signalling via protein tyrosine kinases and G-protein-coupled signalling. The factors which influence HIV-1 replication and the intracellular signalling mechanisms elicited by the virus are not well understood. In this study, it was demonstrated that exposure of peripheral blood lymphocytes (PBLs) to a T-cell tropic strain of HIV-1 evokes signal(s) which results in downregulation of intracellular cAMP. In addition, pre-incubation of PBLs with the Gi-protein inhibitor Pertussis toxin mediated a significant inhibition of HIV-1 replication. These data strongly suggest that HIV-1 employs CD4 receptors and Gi-coupled proteins for entry into target cells and that productive HIV-1 infection is dependent on an active signalling event.

Integrin signalling defects in T-lymphocytes in systemic lupus erythematosus.

OBJECTIVE: To establish the relationship between T cell responses to integrin coreceptor stimulation and B cell hyperreactivity as measured by pathologic autoantibody production. METHODS: Peripheral blood mononuclear cells from 42 patients with SLE according to the American Rheumatism Association criteria were examined for their ability to adhere to plate-immobilised fibronectin. Co-stimulation assays were performed on the same cells using anti-CD3 antibody alone or co-immobilised with an anti-beta1-integrin antibody. Proliferative responses were measured by 3[H]thymidine pulsing on day 3 and activation was determined using a commercial protein kinase C assay, the protocol being established by our group in association with Promega. Beta-integrin expression was established by FACS analysis. RESULTS: An impaired PKC response to integrin-mediated activation was found in T-lymphocytes from 6/21 (29%) SLE patients, which correlated significantly with an absence of anti-dsDNA antibody in patient sera, irrespective of prednisolone treatment. Integrin co-stimulation of TcR/CD3-induced proliferation and T cell adhesion to fibronectin were also impaired among 5/21 (24%) and 6/15 (40%) patients studied, respectively. CONCLUSION: We hypothesise that the integrity of beta1-integrin signalling pathways may influence pathological antibody production in SLE by affecting T-lymphocyte activation and interactions between T- and B-lymphocytes.