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ABSTRACTThe genetic background and the presence of several virulence factors of Staphylococcus aureusisolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus(MSSA) and 7 methicillin-resistant S. aureus(MRSA) isolates were collected. The genetic background was determined by spatyping and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec(SCCmec) typing was performed. Among the MSSA isolates, 313 different spatypes were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmectype), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureusgenetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.

ABSTRACTWe describe an improved multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for genotyping Staphylococcus aureus. We compare its performance to those of multilocus sequence typing (MLST) and spatyping in a survey of 309 strains. This collection includes 87 epidemic methicillin-resistant S. aureus(MRSA) strains of the Harmony collection, 75 clinical strains representing the major MLST clonal complexes (CCs) (50 methicillin-sensitive S. aureus[MSSA] and 25 MRSA), 135 nasal carriage strains (133 MSSA and 2 MRSA), and 13 published S. aureusgenome sequences. The results show excellent concordance between the techniques' results and demonstrate that the discriminatory power of MLVA is higher than those of both MLST and spatyping. Two hundred forty-two genotypes are discriminated with 14 VNTR loci (diversity index, 0.9965; 95% confidence interval, 0.9947 to 0.9984). Using a cutoff value of 45%, 21 clusters are observed, corresponding to the CCs previously defined by MLST. The variability of the different tandem repeats allows epidemiological studies, as well as follow-up of the evolution of CCs and the identification of potential ancestors. The 14 loci can conveniently be analyzed in two steps, based upon a first-line simplified assay comprising a subset of 10 loci (panel 1) and a second subset of 4 loci (panel 2) that provides higher resolution when needed. In conclusion, the MLVA scheme proposed here, in combination with available on-line genotyping databases (including http://mlva.u-psud.fr/), multiplexing, and automatic sizing, can provide a basis for almost-real-time large-scale population monitoring of S. aureus.

Staphylococcus aureus can cause a wide variety of infections, ranging from minor skin infections to post-operative wound infections. Its adaptive power to antibiotics has resulted in the emergence of methicillinresistant S. aureus (MRSA) in the beginning of the 1960s. Resistance to methicillin and all other ß-lactam antibiotics is caused by the mecA gene, which is situated on a mobile genomic island, the Staphylococcal Cassette Chromosome mec (SCCmec). Seven main SCCmec types, I to VII, have been distinguished. The most important methods used to study the molecular epidemiology of MRSA are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing and SCCmec typing. These methods have been used to investigate the evolution of the MRSA clones that have emerged since the 1960s, and to study their worldwide dissemination. Early MRSA clones were hospital-associated (HA-MRSA). However, from the late 1990s, community-associated MRSA (CA-MRSA) has emerged. CA-MRSA harbors SCCmec type IV, V or VII, has a genetic background that is often distinct from HA-MRSA, and is often associated with the toxin Panton- Valentine leukocidin (PVL). However, the distinction between HA-MRSA and CA-MRSA is beginning to blur, and CA-MRSA is endemic in many US hospitals nowadays. This review describes the latest developments concerning the structure of SCCmec, the methods used to investigate the molecular epidemiology of MRSA, the molecular evolution of MRSA as well as the major challenges that are awaiting researchers in the near future.

Hospital-acquired infections (HAI) increase morbidity and mortality and constitute a high financial burden on health care systems. An effective weapon against HAI is early detection of potential outbreaks and sources of contamination. Such monitoring requires microbial typing with sufficient reproducibility and discriminatory power. Here, a microbial-typing method is presented, based on Raman spectroscopy. This technique provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Although the method is generally applicable, we used 118 Staphylococcus aureusisolates to illustrate that the discriminatory power matches that of established genotyping techniques (numerical index of diversity [D] = 0.989) and that concordance with the gold standard (pulsed-field gel electrophoresis) is high (95%). The Raman clustering of isolates was reproducible to the strain level for five independent cultures, despite the various culture times from 18 h to 24 h. Furthermore, this technique was able to classify stored (-80°C) and recent isolates of a methicillin-resistant Staphylococcus aureus-colonized individual during surveillance studies and did so days earlier than established genotyping techniques did. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratory settings. This will set the stage for continuous, automated, real-time epidemiological monitoring of bacterial infections in a hospital, which can then be followed by timely corrective action by infection prevention teams.

ABSTRACTWe observed that, between 1999 and 2006, up to 50% of the methicillin-susceptible Staphylococcus aureus(MSSA) bloodstream isolates in our hospital had a genetic background common to endemic methicillin-resistant S. aureusclones (clonal complex 5 [CC5], CC8, CC22, CC30, and CC45). Furthermore, several successful MSSA lineages, such as CC7 and CC15, were observed.

Objectives: The objectives of this study were (i) to investigate the genetic background of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from Croatia and (ii) to monitor the prevalence of Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) among these isolates.Methods: Eighty-two hospital-acquired MRSA bloodstream isolates, collected in 2001 and 2002 in Croatia, were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). The presence of genes encoding PVL and TSST-1 was investigated by real-time PCR.Results: All strains were multiresistant and were distributed among 16 different similarity groups as determined by PFGE. Two of the groups, groups H and K, harboured the majority of the MRSA strains with 52 and 12%, respectively. The predominant SCCmec type found among the isolates was type I (89%). Eleven per cent of the strains harboured a modified SCCmec type III, which contained, in contrast to the regular type III, an additional dcs region. One strain harboured a novel SCCmec type, containing the ccrC gene in combination with the mecI gene, the dcs region, the locus between pI258 and Tn554 (locus E) and the locus between Tn554 and orfX (locus F). MLST showed the presence of ST111-MRSA-I and ST247-MRSA-I among Croatian MRSA isolates. All isolates were negative for both PVL and TSST-1.Conclusions: These results indicate the emergence of ST111-MRSA-I and ST247-MRSA-I in Croatia among MRSA bloodstream isolates. The virulence factors PVL and TSST-1 were not present among these isolates.

ABSTRACTThe Euregio Meuse-Rhine (EMR) is formed by the border regions of Belgium, Germany, and The Netherlands. Cross-border health care requires infection control measures, in particular since the prevalence of methicillin-resistant Staphylococcus aureus(MRSA) differs among the three countries. To investigate the dissemination of MRSA in the EMR, 152 MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), SCCmectyping, and multilocus sequence typing. PFGE revealed major clonal groups A, G, L, and Q, suggesting dissemination of MRSA in the EMR. Group A harbored mainly SCCmectype III and sequence types (STs) 239 and 241. The majority of the strains from group G harbored SCCmectype I and ST8 and ST247, whereas most strains from group L carried either SCCmectype IV or type I. Within group L, ST8 and ST228 were found, belonging to clonal complexes 8 and 5, respectively. Most strains from group Q included SCCmectype II and were sequence typed as ST225. Both ST225-MRSA-II and ST241-MRSA-III were novel findings in Germany. In addition, the SCCmectype of two isolates has not been described previously. One strain was classified as SCCmectype III but harbored the plsgene and the dcsregion. Another strain was characterized as SCCmectype IV but lacked the dcsregion. In addition, one isolate harbored both SCCmectype V and Panton-Valentine leukocidin. Finally, the SCCmectype of the strains was found to be correlated with the antibiotic susceptibility pattern.

AbstractThe Severe Acute Respiratory Syndrome (SARS) has been dubbed as an epidemic of fear. A public education campaign was launched in Singapore to allay fears and encourage the adoption of appropriate behaviour. In a survey conducted in May 2003, the anxiety level of respondents during the SARS outbreak and their satisfaction with media messages were assessed. Possible implications for behaviour were also investigated. While over half of the respondents were anxious about the SARS situation, 87 per cent reported that they found the SARS information provided was accurate, timely, complete and helpful. However, satisfaction about media information was not associated with any positive or negative behaviour of respondents. Respondents who had a higher level of anxiety towards neighbours being quarantined and the spread of SARS to the community tended to be people who practised cautious behaviour towards SARS or adopted appropriate personal hygiene and healthy lifestyle habits. A high anxiety level towards seeking medical help at general practitioner (GP) clinics or government polyclinics is related to high social responsibility, defined by the public education campaign as the wearing of masks when one was unwell and the regular checking of body temperature.

BIA is easy, non-invasive, relatively inexpensive and can be performed in almost any subject because it is portable. Part II of these ESPEN guidelines reports results for fat-free mass (FFM), body fat (BF), body cell mass (BCM), total body water (TBW), extracellular water (ECW) and intracellular water (ICW) from various studies in healthy and ill subjects. The data suggests that BIA works well in healthy subjects and in patients with stable water and electrolytes balance with a validated BIA equation that is appropriate with regard to age, sex and race. Clinical use of BIA in subjects at extremes of BMI ranges or with abnormal hydration cannot be recommended for routine assessment of patients until further validation has proven for BIA algorithm to be accurate in such conditions. Multi-frequency- and segmental-BIA may have advantages over single-frequency BIA in these conditions, but further validation is necessary. Longitudinal follow-up of body composition by BIA is possible in subjects with BMI 16–34kg/m2without abnormal hydration, but must be interpreted with caution. Further validation of BIA is necessary to understand the mechanisms for the changes observed in acute illness, altered fat/lean mass ratios, extreme heights and body shape abnormalities.

We investigated the association of C1431T and Pro12Ala polymorphisms at the peroxisome proliferator-activated receptor γ (PPARγ) locus with plasma lipids and insulin resistance-related variables, according to diabetes status, in a large and representative Asian population from Singapore consisting of 2,730 Chinese, 740 Malays, and 568 Indians. Moreover, we estimated the diabetes risk and examined gene-nutrient interactions between these variants and the ratio of polyunsaturated fatty acid to saturated fat (SFA) in determining body mass index (BMI) and fasting insulin. We found differential effects of these gene variants. The Pro12Ala polymorphism was more associated with plasma lipids and fasting glucose concentrations, whereas the C1431T polymorphism was related to the risk of diabetes. Carriers of the 12Ala allele had higher HDL-cholesterol than did Pro12Pro homozygotes (P< 0.05), and the effect of the 12Ala allele on fasting glucose was modified by diabetes status (P< 0.001). After controlling for confounders, carriers of the T allele had decreased risk of diabetes compared with CC homozygotes [odds ratio (OR) 0.73, 95% confidence interval (CI) 0.58–0.93; P= 0.011]; this effect was stronger in Indians (OR 0.38, 95% CI 0.15–0.92; P= 0.032).

AbstractMarch 2003 marked the start of the Severe Acute Respiratory Syndrome (SARS) outbreak in Singapore. A public education campaign to prevent and contain the spread of the virus and allay the fear of the residents was put in place. A household survey was conducted to evaluate this public education programme. The survey assessed public knowledge, attitude and behaviour, identified concerns and provided feedback on measures undertaken by the government.About 94 per cent of the respondents could cite fever as a symptom for SARS. For precautions against SARS, 87 per cent practised at least one measure. More than 63 per cent were most concerned for themselves or their loved ones being infected with SARS. More than 53 per cent of the respondents were anxious to seek medical attention at Tan Tock Seng Hospital (TTSH), the designated hospital for SARS in Singapore, and 82 per cent reported that they were confident in TTSH's ability to deal with the virus. The public was more cautious about their behaviour during the outbreak, with 3 per cent failing to adopt any of the behavioural changes. About 93 per cent were satisfied or very satisfied with the government's response to SARS.Continuous education of the public to be vigilant and to adopt appropriate personal hygiene and practise healthy lifestyles and socially responsible habits to guard against SARS is needed.

Abstract: The objectives of this study were to obtain values for resting metabolic rate in Italians in relation to parameters of body composition, and to compare them to predicted values using the FAO/WHO/UNU equation. We performed a cross-sectional observational study of 131 healthy subjects (46 males and 85 females) at the Human Nutrition Unit, University Tor Vergata, Rome. Body composition was determined by dual energy X-ray absorptiometry (DXA) and resting metabolic rate was calculated using their Weir formula. Resting metabolic rate was 1865 ± 234 kcal/day in males and 1354 ± 154 kcal/day in females. These values decreased slightly with age. The relationships with weight and age were stronger than that with lean mass from DXA as independent variables in multiple regression analysis. Mean resting metabolic rates predicted with FAO/WHO/UNU and Harris-Benedict formula were not significantly different from measured values except for the Harris-Benedict value for females (p < 0.01). Individual differences between measured and predicted values were notably high. The measured values were higher than those reported in the literature. The prediction of resting metabolic rate is more accurate with simple anthropometric parameters than with fat-free mass obtained by DXA. The individual error in the predicted values can be so high that for individual use a measured value is preferred over an estimated value.

Fat-free mass hydration, the ratio of total body water (TBW) to fat-free mass (FFM), is stable at ?0.73 in mammals, and this constancy provides a means of estimating total body fat in vivo. As there is no mechanistic theory to explain the magnitude and variability in TBWFFM, the present investigation describes a cellular-level model indicating that FFM hydration is determined by four factors: body cell mass hydration, extracellular fluid hydration, ratio of extracellular solids to TBW, and ratio of extracellular water to intracellular water. According to the model, TBWFFM can be predicted for adult humans as a mean of 0.73, and a variation range 0.69-0.77. The suggested modeling approach provides a conceptual framework for TBW-fat estimation method and identifies important areas that remain to be studied.

The effects of an episode of acute classical dengue fever on extracellular water (ECW), intracellular water (ICW), and total body water (TBW) were measured in nine patients using conventional dilution techniques; and the findings were compared with the outcome variables from whole body impedance spectroscopy (BIS), extracellular fluid resistance (Recf), and intracellular fluid resistance (Ricf). The patients were assessed on admission with febrile presentation (DI), at discharge after the defervescence of the fever cycle at about five days postadmission (DII), and seven days thereafter (DIII). As a reference group, 15 persons without acute or chronic illness were enrolled. Total body water was unaltered during the course of disease and was not different from that in normal healthy subjects. However, body water shifted from the intracellular to the extracellular compartment in patients from the acute phase to convalescence, as reflected in the ratios of ECWTBW and ECWICW. These ratios were significantly higher in convalescent dengue patients (DIII) than in the reference group ( p< 0.05 ). Increasing ECW, from the acute phase of the disease to convalescence, was associated with a significant decrease in Recf (719 ± 95, 693 ± 89, 643 ± 81 ?; p< 0.0001 ) and in RecfRicf ( p< 0.01 ). Recf and RecfRicf were higher in the acute phase (DI) of dengue fever compared to controls ( p< 0.05 ). We conclude that dengue fever is characterized by a relative expansion of ECW during the course of disease and convalescence. BIS was sensitive in determining the hydrational profile in dengue fever patients.

Fat is a normal component of the human body that is stored in adipose tissue. Obesity can be defined as a condition of excessive fat accumulation to the extent that health and well-being are affected. Body fat can be determined in vivo in different ways, using rather accurate laboratory techniques or using simple estimation techniques that can also be applied in field conditions. For population studies, the World Health Organization defines cut-off values for obesity based on the body mass index (BMI): weight/height squared (kg/m2). Generally, for adults, if the BMI exceeds 25 kg/m2, a subject is considered to be overweight, and if the BMI exceeds the value of 30 kg/m2, a subject is considered obese. However, the relationship between body fat percentage and BMI differs between ethnic groups, and, as a consequence, cut-off points for overweight and obesity based on BMI will have to be ethnicity specific. This means that, in some populations, the cut-off points could be lower or higher than the recommended figures. Adapting cut-off point values will have important consequences for prevalence data in some countries as the prevalence of obesity will dramatically increase or decrease. The prevalence of obesity, in children as well as in adults, is high in many countries all over the world and is rising. Given the impact of obesity on health, this is a public health issue that needs to be addressed seriously.

Although it is often assumed that skeletal muscle (SM) is a fixed proportion of adipose tissue-free body mass (ATFM), there is little data in living humans that supports this hypothesis. Limited data from elderly cadavers suggests a relatively constant proportion of ATFM as SM in men (mean ± SD, 0.540 ± 0.046) and women (0.489 ± 0.049). The present study tested the hypothesis that SM is a relatively constant proportion of ATFM in healthy men. Whole body SM and ATFM were measured using multiscan computerized axial tomography. The SM/ATFM ratio in the men (n = 24) was 0.563 ± 0.036 which was not significantly different from the value found in the elderly male cadavers. Multiple regression analysis indicated that the SM/ATFM ratio was significantly related to 1/ATFM (P = 0.036) and the body mass index (P = 0.024), and not with age. Further study is needed of other possible factors such as physical activity, gender, race, and disease state, that may effect the relationship between SM and ATFM. Am. J. Hum. Biol. 9:487–492, 1997. © 1997 Wiley-Liss, Inc.

OBJECTIVES: Multi‐Frequency Bioelectrical Impedance Analysis (MFBIA) is a quick, simple, and inexpensive method to assess body fluid compartments. This study aimed at determining the validity of MFBIA in detecting clinically relevant changes of fluid balance in geriatric patients.

Polysomes from cross-, self- and unpollinated ovaries have been injected in Xenopus laevis eggcells. The synthesised proteins have been identified by means of gel-electrophoresis. It appeared that few hours after pollination the protein pattern of the pollinated ovary has changed in comparison with that of unpollinated ones. However, in this early stage, there is only little difference found in the protein pattern of selfed and crossed ovaries. From 12 h after pollination and onwards, the difference between pollinated and unpollinated ovaries becomes very obvious, as does also the difference between crossed and selfed ovaries. At 24 h after pollination many different proteins exist in crossed and selfed ovaries. From 32 h to 48 h after pollination the crossed ovaries differentiate further on, whereas the ovaries after self-pollination show more and more proteins which were also found in the unpollinated ovaries of that age. Possible explanations and hypothesis for these phenomena are discussed as well as the probable occurrence of signal(s) arriving from the stigma or the style at the ovary.

Urinary excretion of caffeine in two populations (men and women) of cyclotourists was measured, at rest and during exercise, after oral administration of 350 mg of caffeine in aqueous solution. The so-called “total metabolites”, as measured by the EMIT test, were also determined, as well as urinary creatinine. At rest, elimination in relation to body weight was identical in men and women. During exercise a fivefold decrease in the female and twofold decrease in the male populations were observed. After exercise, caffeine elimination was greater than during the physical trial but remained lower for women than for men. “Total metabolites” excretion showed evidence for a slowing of caffeine catabolism during exercise and a restart of it after exercise. The caffeine content of beverages varies considerably from one country to another, depending on local customs, so that caffeine intake may be highly variable. Our results lead us to query the validity of the upper authorized official limit for urinary caffeine (12 μg·ml−1) in doping controls. The nature of the sporting event, sex, weight, and sampling delay after exercise are all factors that argue against the utilization of a unique standard.

From in-vitro protein synthesis studies it has been shown that in Petunia hybrida (clone W166K, incompatibility genes S1S2) the ovary is stimulated to synthesize proteins before the pollentubes reach the ovary. A difference in protein metabolism is detected after self- and cross-pollination. On the basis of this result has been concluded that a signal has to be sent from the stigma or the style towards the ovary which induces the changes in metabolic activity. The signal must be different for cross- and self-pollination.

Risk factors for breast cancer were compared in 107 women with interval breast cancer (cancers occurring within 2 years after a negative screen) and 258 women with breast cancer detected at 1st screening. All women (aged 40–67) were screened in the DOM project (the Utrecht programme for the early detection of breast cancer). Women with an interval cancer reported more often a history of benign breast disease (OR 4.66, 95% C.I. 2.08-10.41) and an artificial menopause (OR 4.07; 95% C.I. 1.74-9.55) than women with a screen detected cancer. Women with an interval cancer were taller than women with a screen detected cancer; this was seen most clearly in women with an artificial menopause (X2 for trend = 5.88; p = 0.02) and to a lesser extent in premenopausal women (X2 for trend = 1.70; p = 0.19). Premenopausal women with an interval cancer were heavier than women with a screen detected cancer (X2 for trend = 4.66; p = 0.03); whereas natural postmenopausal women with an interval cancer were leaner than women with a screen detected cancer (X2 for trend = 1.57; p = 0.21). All analyses were done while correcting for age and selected other risk factors for breast cancer.

The relationship between the fatty acid composition of subcutaneous adipose tissue and diet was estimated in 59 Dutch women aged 32-35 years. Food consumption was estimated by taking the means of nineteen 24-hour recalls administered over a period of two and a half years, August 1981-December 1983. Highly significant correlations were found between linoleic acid content of fat tissue and diet (r = 0.70) and also between the linoleic acid-to-saturated fatty acid (linoleic/S) ratio of fat tissue and diet (r = 0.62). This confirms the hypothesis that on an individual level the fatty acid composition of the adipose tissue is a valid index for the habitual dietary fatty acid composition of free-living adults. When using one 24-hour recall instead of the average of 19 recalls, the correlation coefficient between the linoleic/S ratio of the diet and that of the adipose tissue was substantially decreased. This demonstrates the weakening effect of the large day-to-day variation in within-person intake on the correlation between a short-term assessment of the nutrient intake of an individual and a biochemical indicator of long-term nutritional status.