Whole-Genome Sequencing Reveals the Contribution of Long-Term Carriers in Staphylococcus aureus Outbreak Investigation.

Whole-genome sequencing (WGS) makes it possible to determine the relatedness of bacterial isolates at a high resolution, thereby helping to characterize outbreaks. However, for Staphylococcus aureus, the accumulation of within-host diversity during carriage might limit the interpretation of sequencing data. In this study, we hypothesized the converse, namely, that within-host diversity can in fact be exploited to reveal the involvement of long-term carriers (LTCs) in outbreaks. We analyzed WGS data from 20 historical outbreaks and applied phylogenetic methods to assess genetic relatedness and to estimate the time to most recent common ancestor (TMRCA). The findings were compared with the routine investigation results and epidemiological evidence. Outbreaks with epidemiological evidence for an LTC source had a mean estimated TMRCA (adjusted for outbreak duration) of 243 days (95% highest posterior density interval [HPD], 143 to 343 days) compared with 55 days (95% HPD, 28 to 81 days) for outbreaks lacking epidemiological evidence for an LTC (P = 0.004). A threshold of 156 days predicted LTC involvement with a sensitivity of 0.875 and a specificity of 1. We also found 6/20 outbreaks included isolates with differing antimicrobial susceptibility profiles; however, these had only modestly increased pairwise diversity (mean 17.5 single nucleotide variants [SNVs] [95% confidence interval {CI}, 17.3 to 17.8]) compared with isolates with identical antibiograms (12.7 SNVs [95% CI, 12.5 to 12.8]) (P < 0.0001). Additionally, for 2 outbreaks, WGS identified 1 or more isolates that were genetically distinct despite having the outbreak pulsed-field gel electrophoresis (PFGE) pulsotype. The duration-adjusted TMRCA allowed the involvement of LTCs in outbreaks to be identified and could be used to decide whether screening for long-term carriage (e.g., in health care workers) is warranted. Requiring identical antibiograms to trigger investigation could miss important contributors to outbreaks.

A two-stage algorithm for Clostridium difficile including PCR: can we replace the toxin EIA?

A two step, three-test algorithm for Clostridium difficile infection (CDI) was reviewed. Stool samples were tested by enzyme immunoassays for C. difficile common antigen glutamate dehydrogenase (G) and toxin A/B (T). Samples with discordant results were tested by polymerase chain reaction detecting the toxin B gene (P). The algorithm quickly identified patients with detectable toxin A/B, whereas a large group of patients excreting toxigenic C. difficile but with toxin A/B production below detection level (G(+)T(-)P(+)) was identified separately. The average white blood cell count in patients with a G(+)T(+) result was higher than in those with a G(+)T(-)P(+) result.

A cluster of Listeria monocytogenes infections in hospitalised adults, Midlands, England, February 2011.

Hospital-acquired listeriosis cases are not commonly reported but remain a significant public health problem. We report on three cases in patients with underlying conditions occurring during one week in February 2011. The cases had common exposure to pre-packed sandwiches and salads manufactured in compliance with regulations. Breaches in cold chain and shelf life controls at hospital level were identified as key contributing factors. Rigorous hospital food management systems remain important for patient safety.

Community and nosocomial transmission of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus: implications for healthcare.

In the UK, infections due to Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus (PVL-MRSA) have been reported sporadically. In September 2006, a fatal PVL-MRSA infection occurred in a Filipino healthcare worker (HCW) after she underwent caesarean section. Throat and nasal swabs were obtained from contacts of cases in community and hospital. MRSA with an antibiogram similar to the PVL-MRSA strain were characterised including toxin gene profiling, polymerase chain reaction- and sequence-based typing. Carriers underwent decolonisation treatment, and HCWs were restricted from patient care until they and their household members were considered negative for PVL-MRSA. The PVL-MRSA belonged to ST30, was protein A gene (spa) type t019, SCCmec IVc, agr 3, and resistant only to beta-lactam antibiotics. Representatives of the same lineage were identified among a further 16 individuals in community and hospital. Infections likely to be caused by PVL-MRSA had occurred in 12 cases, and were likely to be hospital-acquired in two patients (one fatal) and occupationally acquired in one HCW. Nine cases worked as nursing staff in the hospital. Eight of these had emigrated from the Philippines in the previous five years and were linked socially. Thus, PVL-MRSA-ST30 was detected in a HCW community in the UK. This is the first report of nosocomial transmission of this pandemic clone in the UK associated with a fatality. Increased vigilance in healthcare and community is needed in response to this emerging threat.

Single-dose cefuroxime with gentamicin reduces Clostridium difficile-associated disease in hip-fracture patients.

Antibiotic-associated Clostridium difficile diarrhoea may complicate recovery from surgery for proximal femoral fracture. We undertook a four-year case-control study to evaluate a change in antibiotic prophylaxis in our department. During the period January 2003 to January 2005, patients received three doses of prophylactic cefuroxime (1.5g). We then introduced a new regimen, comprising of one single dose of cefuroxime (1.5g) with gentamicin (240mg) at induction. Prior to the change in prophylaxis, 912 patients underwent surgery for neck of femur fracture, and from March 2005 to March 2007, 899 patients had surgery under the new regimen. Thirty-eight patients developed C. difficile infection (4.2%) in the initial group, compared with 14 patients (1.6%) in the group with the new regimen (P=0.009). The incidence of C. difficile infection increased throughout the rest of the hospital over the same time period. Patients with C. difficile infection had a statistically significant increase in antibiotic exposure, inpatient stay, morbidity and inpatient mortality. The main challenges regarding prophylactic antibiotic selection are infection due to meticillin-resistant Staphylococcus aureus (MRSA) and C. difficile-associated diarrhoea. We advocate the use of the new regimen as an alternative to multiple-dose cephalosporin antibiotics for the prevention of C. difficile infection in this group of high-risk patients.

An outbreak of Escherichia coli O157 gastroenteritis in a care home for the elderly.

In the summer of 2001 an outbreak of Escherichia coli O157 gastroenteritis affected staff and residents of a care home for the elderly in the West Midlands, UK. E. coli O157 phage type 2 was isolated from faeces in eight patients and 12 staff members. Thirty-five staff and 40 residents met the case definition for clinical gastrointestinal infection. Serological testing identified a further 14 possible cases of infection amongst asymptomatic staff and residents. The outbreak was atypical, as the disease seemed to be milder than has been observed in past outbreaks in similar settings. The index case, a member of staff, developed bloody diarrhoea and haemolytic-uraemic syndrome (HUS), but only one resident developed bloody diarrhoea and required hospitalization. No deaths occurred, despite the high-risk nature of the affected population. The source of the outbreak could not be identified. The prolonged nature of the outbreak and observed lapses in infection control practices indicated that person-to-person spread was the likely route of transmission. This outbreak illustrates the importance of observing appropriate infection control measures in the institutions providing residential and nursing care to the elderly.

Infective discitis with Neisseria sicca/subflava in a previously healthy adult.

STUDY DESIGN: A case report of Neisseria sicca/subflava discitis in a healthy elderly female. OBJECTIVE: To report a rare case, which is usually seen exclusively in children. SETTING: Stoke on Trent, England. METHOD: Case report, a 65-year-old female with a 6 month history of back and bilateral leg pain. X-rays showed collapse of L4/5 disc. No neurological deficit. Magnetic resonance imaging supported the clinical suspicion of discitis. Percutaneous biopsy followed 2 weeks later by open biopsy with bilateral root decompression was performed. Culture of L4/5 disc tissue produced Neisseria sicca/subflava. The patient was treated with a 4-week course of intravenous amoxycillin. Follow-up at 3 months confirmed clinical resolution of original symptoms. CONCLUSION: Any organism cultured from biopsy needs to be interpreted within the context of the clinical case. If clinical suspicion is high, further weight must be added to the finding of unusual or environmental organisms and culture of a repeat aspirate or biopsy may clarify the significance.

Hydroxyurea interferes with antigen-dependent T-cell activation in vitro.

BACKGROUND: Hydroxyurea is believed to inhibit human immunodeficiency virus type 1 (HIV-1) in HIV disease by decreasing the amount of intracellular deoxynucleotides needed for viral replication. A plasma concentration of 400 micromol L-1 is tolerated in oncological diseases. The present study focused on the possible interference of hydroxyurea with antigen-dependent T-cell activation as an alternative explanation for inhibiting HIV replication in vivo. METHODS: The effect of hydroxyurea on common antigen-induced cell proliferation was studied in peripheral blood mononuclear cells (PBMC) in vitro. RESULTS: Hydroxyurea inhibited Candida albicans-induced cell proliferation at a low concentration (1 micromol L-1), while at least 10 micromol L-1 was required to block HIV-1 replication in phytohaemagglutinin (PHA)-stimulated PBMC. CONCLUSION: Hydroxyurea inhibits antigen-induced lymphoproliferation in vitro at a concentration at which it does not inhibit PHA-induced HIV replication. Hydroxyurea may inhibit HIV-1 in CD4+ T cells in vivo not only by decreasing the amount of intracellular deoxynucleotides, but more specifically by interfering with antigen-dependent T-cell activation, thereby causing a reduction in the number of HIV target cells.

Activation and cell cycle antigens in CD4+ and CD8+ T cells correlate with plasma human immunodeficiency virus (HIV-1) RNA level in HIV-1 infection.

The relationship between T cell activation and human immunodeficiency virus type 1 (HIV-1) replication was studied in HIV-infected subjects, 20 with and 10 without anti-HIV treatment. Expression of Ki-67 proliferation-associated antigen was increased in CD4+ and CD8+ T cells and correlated with HLA-DR. In subjects without anti-HIV treatment, the plasma HIV-1 RNA level correlated with HLA-DR in CD4+ T cells, with Ki-67 in CD8+ T cells, and with expression of CD38 in both T cell subsets. A proportion of treated subjects had increased T cell activation despite 4 months of highly active antiretroviral treatment (HAART). In subjects receiving HAART, a high percentage of HLA-DR+ CD4+ T cells was associated with signs of opportunistic infections. This work supports the concept that, in the natural course of HIV-1 infection, HIV replication itself leads to general T cell activation and that opportunistic infections generate additional CD4+ T cell activation and HIV replication.

[Vertical HIV-I-transmission. I. Risk and prevention in pregnancy]. / Verticale HIV-I-transmissie. I. Risico en preventie bij de zwangere.

Without anti-HIV treatment, mother to child HIV-I transmission occurs in 15-30% of HIV positive pregnancies. Transmission occurs mostly in the last trimester or at birth. The maternal virus load in the last trimester and around birth is strongly related to the risk of HIV transmission to the child. This risk can be reduced during pregnancy by anti-HIV treatment and in certain cases by performing a caesarean section. It is recommended to determine the plasma virus load several times during pregnancy. If the virus load is found to be high, measurement of plasma anti-HIV drug concentrations and anti-HIV drug resistance may prompt modification of the anti-HIV drug regimen with the objective of achieving maximal suppression of virus replication in the last trimester.

[Vertical HIV-I-transmission. II. HIV-diagnosis in a child]. / Verticale HIV-I-transmissie. II. HIV-diagnostiek bij het kind.

In newborn children from HIV-infected women early establishment of HIV infection is of importance for optimal therapy of HIV-infected children and avoidance of unnecessary medication in uninfected children. A more than 95% reliable diagnosis of HIV infection can now be obtained at the age of four weeks by polymerase chain reaction (PCR) technology. Before this age a positive PCR result is relevant since it necessitates additional investigation such as measuring anti-HIV drug resistance and may lead to modification of anti-HIV treatment. Prophylaxis against Pneumocystis carinii is not needed if HIV infection can not be demonstrated by PCR after the age of four weeks.

Mannoproteins of Cryptococcus neoformans induce proliferative response in human peripheral blood mononuclear cells (PBMC) and enhance HIV-1 replication.

To investigate the possible role of Cryptococcus neoformans var. neoformans in HIV disease progression, and to identify the responsible cryptococcal components, an in vitro cell culture model was set up to study the C. neoformans-induced enhancement of HIV replication in HIV-1-infected PBMC. Similar to whole C. neoformans, cell-wall membrane fraction and mannoproteins induced proliferation of PBMC and enhancement of lymphotropic HIV replication in HIV-infected PBMC, while galactoxylomannan did not. MoAbs capable of interfering with MHC class II-mediated antigen presentation prevented the induction of cell proliferation by whole C. neoformans or cryptococcal mannoproteins. MoAb binding to adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) also inhibited C. neoformans-induced cell proliferation. In addition, anti-MHC class II MoAb inhibited the enhancement of HIV replication by C. neoformans. The results suggest that: (i) C. neoformans may accelerate HIV disease progression by stimulation of HIV replication through MHC class II-mediated antigen presentation; and (ii) cryptococcal mannoprotein may be one of the responsible components. The ability to enhance HIV replication in PBMC in vitro is not unique for C. neoformans. However, this is the first report to study in detail a yeast-induced enhancement of HIV replication in PBMC.

Functional magnetic resonance imaging studies of emotional processing in normal and depressed patients: effects of venlafaxine.

BACKGROUND: Functional magnetic resonance imaging (fMRI) techniques were used to identify the neural circuitry underlying emotional processing in control and depressed subjects. Depressed subjects were studied before and after treatment with venlafaxine. This new technique provides a method to noninvasively image regional brain function with unprecedented spatial and temporal resolution. METHOD: Echo-planar imaging was used to acquire whole brain images while subjects viewed positively and negatively valenced visual stimuli. Two control subjects and two depressed subjects who met DSM-IV criteria for major depression were scanned at baseline and 2 weeks later. Depressed subjects were treated with venlafaxine after the baseline scan. RESULTS: Preliminary results from this ongoing study revealed three interesting trends in the data. Both depressed patients demonstrated considerable symptomatic improvement at the time of the second scan. Across control and depressed subjects, the negative compared with the positive pictures elicited greater global activation. In both groups, activation induced by the negative pictures decreased from the baseline scan to the 2-week scan. This decrease in activation was also present in the control subjects when they were exposed to the positive pictures. In contrast, when the depressed subjects were presented with the positive pictures they showed no activation at baseline, whereas after 2 weeks of treatment an area of activation emerged in right secondary visual cortex. CONCLUSION: While preliminary, these results demonstrate the power of using fMRI to study emotional processes in normal and depressed subjects and to examine mechanisms of action of antidepressant drugs.

A Developmental Functional MRI Study of Prefrontal Activation during Performance of a Go-No-Go Task.

This study examines important developmental differences in patterns of activation in the prefrontal cortex during performance of a Go-No-Go paradigm using functional magnetic resonance imaging (fMRI). Eighteen subjects (9 children and 9 adults) were scanned using gradient echo, echo planar imaging during performance of a response inhibition task. The results suggest four general findings. First, the location of activation in the prefrontal cortex was not different between children and adults, which is similar to our earlier pediatric fMRI results of prefrontal activation during a working memory task (Casey et al., 1995). Second, the volume of activation was significantly greater for children relative to adults. These differences in volume of activation were observed predominantly in the dorsal and lateral prefrontal cortices. Third, although inhibitory processes have typically been associated with more ventral or orbital frontal regions, the current study revealed activation that was distributed across both dorsolateral and orbitofrontal cortices. Finally, consistent with animal and human lesion studies, activity in orbital frontal and anterior cingulate cortices correlated with behavioral performance (i.e., number of false alarms). These results further demonstrate the utility of this methodology in studying pediatric populations.

Enhancement of HIV-1 replication in peripheral blood mononuclear cells by Cryptococcus neoformans is monocyte-dependent but tumour necrosis factor-independent.

OBJECTIVE: To investigate the possible role of Cryptococcus neoformans in HIV-1 pathogenesis. DESIGN: An in vitro system was developed to study HIV-1 replication in freshly HIV-1-infected peripheral blood mononuclear cells (PBMC) incubated with whole azide-killed C. neoformans. METHODS: Human PBMC or peripheral blood lymphocytes were infected with lymphocytotropic HIV-1 and incubated with azide-killed encapsulated or non-encapsulated C. neoformans for 10 days. Viral replication was followed by HIV-1 p24 enzyme-linked immunosorbent assay and median tissue culture infective dose determination. Tumour necrosis factor (TNF) release by PBMC, induced by C. neoformans, was measured. Anti-TNF monoclonal antibodies or pentoxifylline were used to inhibit TNF bioactivity. RESULTS: Both encapsulated and non-encapsulated C. neoformans enhanced HIV-1 replication in PBMC but not in peripheral blood lymphocytes. C. neoformans induced TNF release by PBMC. Inhibition of TNF bioactivity did not block C. neoformans-enhanced HIV-1 replication in PBMC. CONCLUSIONS: C. neoformans can enhance HIV-1 replication in T cells only in the presence of monocytic cells. This enhancement is not dependent on encapsulation nor can it be attributed to TNF release.