Hepatitis C virus (HCV) infection is a frequent problem in hemodialysis units. The prevalence and incidence of HCV infection over a decade were studied in a nephrology unit affected by previous nosocomial HCV transmission. The HCV non-structural 5B protein gene was sequenced to achieve phylogenetic analysis of a new (incident) case of infection. Proportions of patients who were and were not infected with HCV remained similar over the period, as did the inflow and outflow of patients infected previously. In 1997, 12/157 (8%) of patients at the unit (treatment: hemodialysis, peritoneal dialysis, and renal transplant recipients) were positive in HCV RNA, whereas in 2007 the overall number was 9/239 (4%). One patient acquired an HCV infection, and the NS5B sequence in that case clustered with genotype 2b sequences found in patients from an earlier outbreak. Comparing the HCV from the incident patient with several stored longitudinal samples and cloned PCR products from the most likely source patient revealed close phylogenetic relationship with an HCV quasispecies member from the possible source. The source patient and the incident newly infected patient were not scheduled on the same dialysis shift, although the records showed that simultaneous treatment occurred on two occasions during the months preceding transmission. In conclusion, over the 10-year period, the proportion of HCV-infected patients at the unit was unchanged. Only one new infection occurred, which originated from a fellow patient's quasispecies. This establishes phylogenetic analysis as a valuable tool for tracing patient sources of HCV transmission.

Human papillomavirus (HPV) infection is believed to be the central cause of cervical cancer. The viral proteins E6 and E7 from high-risk HPV types prevent cells from differentiating apoptosis and inducing hyperproliferative lesions. Human cervical carcinoma HeLa cells contain integrated human papillomavirus type 18 (HPV-18). Retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HeLa cervical carcinoma cells. Cellular responses to RA are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors. On the other hand, histone deacetylase inhibitors have been shown to be chemopreventive agents for the treatment of cancer cells. In this article, we have examined the antiproliferative effect of RA and histone deacetylase inhibitor BML-210 on HeLa cells, and particularly the effects on protein expression that may be involved in the cell cycle control and apoptosis. Our data suggest that a combination of RA and BML-210 leads to cell growth inhibition with subsequent apoptosis in a treatment time-dependent manner. We confirm that BML-210 alone or in combination with RA causes a marked increase in the level of p21. The changes in the p53 level are under the influence of p38 phosphorylation. We also discovered that the histone deacetylase inhibitor BML-210 causes increased levels of anti-apoptotic protein Bcl-2 and phosphorylated p38 MAP Kinase; the latter link in cell cycle arrest with response to extracellular stimuli. Our results suggest that RA and BML-210 are involved in different signaling pathways that regulate cell cycle arrest and lead to apoptosis of HeLa cells.

Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects.

There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies.

In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear.

We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions.

We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells.

Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.

Dystrobrevins (DBs) bind directly to dystrophin and are prominent components of the dystrophin-associated protein complex (DAPC) that links the cytoskeleton to the extracellular matrix. They are involved in brain development, synapse formation and plasticity, as well as water and ion homeostasis. However, the role of DB in non-muscular cells is not clear. In this study, we show that different alpha-dystrobrevin isoforms are present in promyelocytic leukemia (NB4) cells. Only the biggest alpha-dystrobrevin isoform (DB-alpha), which can be important for its function, was expressed in the membrane fraction of NB4 cells; the other alpha-DB isoforms were found in the hydrophilic cell fractions. Employing the immunoprecipitation and mass spectrometry, we identified novel alpha-DB-interacting proteins involved in cytoskeleton reorganization (actin, tropomyosin, gelsolin, tubulin) and signal transduction process (stathmin, prohibitin, RIBA) during proliferation and differentiation of NB4 cells. Our results suggest that alpha-DB isoforms play a central role in cytoskeleton reorganization via their multiple interactions with actin and actin-associating proteins and may participate in signal transduction process during NB4 cell granulocytic differentiation via directly and non directly associated proteins.

Tetracycline-resistant bacteria and genes encoding tetracycline resistance are common in anthropogenic environments. We studied how wastewater treatment affects the prevalence and concentration of two genes that encode resistance to tetracycline: tetA and tetB. Using real-time PCR we analysed wastewater samples collected monthly for one year at eight key-sites in a full-scale municipal wastewater treatment plant (WWTP). We detected tetA and tetB at each sampling site and the concentration of both genes, expressed per wastewater volume or per total-DNA, decreased over the treatment process. The reduction of tetA and tetB was partly the result of the sedimentation process. The ratio of tetA and tetB, respectively, to total DNA was lower in or after the biological processes. Taken together our data show that tetracycline resistance genes occur throughout the WWTP and that the concentrations are reduced under conventional operational strategies. However, it is not possible to conclude the eventual risk for humans with respect to resistance spreading.

We describe the transmission of hepatitis C virus (HCV) to two patients from a thoracic surgeon who was unaware of his hepatitis C infection. By partial sequencing of the non-structural 5B gene and phylogenetic analysis, the viruses from both patients were found to be closely related to genotype 1a strain from the surgeon. Two further hepatitis C cases were found in relation to the thoracic clinic. Their HCV sequences were related to each other but were of genotype 2b and the source of infection was never revealed. To elucidate the magnitude of the problem, we conducted a prospective study for a period of 17 months in which patients who were about to undergo thoracic surgery were asked to participate. Blood samples were drawn prior to surgery and at least four months later. The postoperative samples were then screened for anti-HCV and, if positive, the initial sample was also analysed. The only two patients (0.4%) identified were confirmed anti-HCV positive before surgery, and none out of 456 evaluable cases seroconverted to anti-HCV during the observation period. Despite the retrospectively identified cases, nosocomial hepatitis C is rare in our thoracic unit. The study points out the risk of transmission of hepatitis C from infected personnel and reiterates the need for universal precautions. © 2008 The Hospital Infection Society.

BACKGROUND: Hepatitis B vaccine has been shown to be highly efficient in preventing hepatitis B. However, 5%-10% of individuals fail to develop protective levels (>or=10 mIU/mL) of antibodies to hepatitis B surface antigen (anti-HBs) and are considered to be nonresponders.

METHODS: A total of 48 nonresponders and 20 subjects naive to the HBV vaccine received a double dose of combined hepatitis A and B vaccine (Twinrix) at 0, 1, and 6 months. The levels of anti-HBs and antibodies to hepatitis A virus (anti-HAV) were determined before vaccination and 1 month after each dose.

RESULTS: Among 44 nonresponders, protective anti-HBs levels were found in 26 (59%) after the first dose and in 42 (95%) after the third dose. Among the control subjects, the corresponding figures were 10% and 100%, respectively. All subjects seroconverted to anti-HAV. The titers of both anti-HBs and anti-HAV were lower in the previously nonresponsive subjects (P< .01).

CONCLUSION: Revaccination of nonresponders to the standard hepatitis B vaccine regimen with a double dose of the combined hepatitis A and B vaccine was highly effective. This is most likely explained by the increased dose, a positive bystander effect conferred by the hepatitis A vaccine, or both.

An assay based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for the measurement of citalopram and its main metabolites desmethylcitalopram and didesmethylcitalopram. The best extraction procedure was performed with end-capped C₂ column utilising secondary silanol interactions to obtain clean extract. The HPLC analysis was done on a phenyl column with a mobile phase without any amine additives. Fluorescence detection gave a limit of detection of 0.8 nmol/l plasma for the compounds analysed.

Objective. Antibiotic resistance is increasing worldwide. The aims of the current study were to determine the fecal carriage of antibiotic-resistant bacteria and antibiotic treatment in surgical patients admitted to hospital due to acute intra-abdominal infections. Materials and methods. Eight Swedish surgical units participated in this prospective multicenter investigation. Rectal swabs were obtained on admission to hospital. Cultures were performed on chromogenic agar and antibiotic susceptibility testing was performed using the disk diffusion method. Extended-spectrum beta-lactamase (ESBL)phenotype was confirmed by Etest. Results. Rectal samples were obtained and analyzed from 208 patients with intra-abdominal surgical infections. Surgery was performed in 134 patients (65%). Cephalosporins were the most frequently used empirical antibiotic therapy. The highest rates of resistance among Enterobacteriaceae were detected for ampicillin (54%), tetracycline (26%), cefuroxime (26%) and trimethoprim-sulfamethoxazole (20%). The prevalence of decreased susceptibility (I + R) for the other antibiotics tested was for ciprofloxacin 20%, piperacillin-tazobactam 17%, cefotaxime 14%, ertapenem 12%, gentamicin 3% and imipenem 0%. ESBL-producing Enterobacteriaceae were found in samples from 10 patients (5%). Three patients had five E. coli isolates producing AmpC enzymes. Conclusions. This study shows a high rate of resistance among Enterobacteriaceae against antibiotics which are commonly used in Sweden and should have implications for the future choice of antibiotics for surgical patients.

The Gram-positive cocci, Staphylococcus aureus, coagulase negative staphylococci (CoNS), Enterococcus faecalis and Enterococcus faecium, are the bacteria most often isolated from patients with hospital acquired infections. S. aureus is one of the most important pathogens and have a variety of virulence mechanisms which help it to infect the patient and cause tissue damage. CoNS and enterococci are low virulent bacteria and predominantly cause infections in individuals with underlying illness, individuals that have undergone surgery or with suppressed immune-system. The aims of this thesis were i) to investigate the susceptibility to different antimicrobial agents among S. aureus, CoNS, E. faecium and E. faecalis isolates from primary care centres, general hospital wards and intensive care units in Denmark, Finland, Norway and Sweden and ii) to study the prevalence of the cytolysin genes and genes involved in biofilm formation among CoNS, E. faecium and E. faecalis. The results in this thesis show that the resistance rates among S. aureus and E. faecalis is still rather low in the north European countries. Among CoNS and E. faecium resistance rates are higher and comparable with rates in other European countries and US. CoNS had statistically significant differences in susceptibility rates between the ward levels with the lower susceptibility rates found at ICUs. Continued surveillance of resistance rates to antimicrobial agents among both staphylococci and enterococci are important internationally, nationally and locally. The results in this thesis also show that all multidrug resistant and 96% of the susceptible CoNS isolates carried at least one of the atlE and aap genes or the ica operon. Among E. faecalis isolates with HLGR, belonging to a cluster of genetically related isolates, both the esp and asa1 genes were carried in a high degree while the cyl operon was less frequently found. In addition, about 30% of unique E. faecalis isolates carried two or more of the virulence genes. Among E. faecium isolates the esp gene was common but asa1 and the cyl operon was not found in any of the isolates. Both CoNS and E. faecalis isolates from hospitalised patients are well equipped with genes involved in biofilm formation. These genes, when expressed and even more in combination with resistance to antimicrobial agents, might give these isolates an advantage compared to other isolates when it comes to adhesion to artificial surfaces, persistence in the hospital environment, colonisation of hospitalised patients and to cause nosocomial infections. Further studies are needed to be able to determine which isolates that causes hospital acquired infections and to evaluate the importance of the genes involved in biofilm formation as virulence factors and about how to prevent biofilm related infections from emerging

The aims of the present study were to identify clinical isolates of multidrug resistant (MDR) and susceptible coagulase negative staphylococci (CoNS), (n=76) to the species level by rpoB amplicon sequencing and to detect and compare the presence of the atlE and aap genes and the ica operon between MDR (n=26) and susceptible (n=27) Staphylococcus epidermidis isolates. Detection of the atlE and aap genes and ica operon was carried out using PCR amplification. Most of the isolates were S. epidermidis, both among the MDR and susceptible CoNS. Staphylococcus haemolyticus was the only other species found in the MDR group. All MDR and 96% of the susceptible S. epidermidis isolates carried the atlE gene. The ica operon was present in about 30% of both the MDR and susceptible S. epidermidis isolates. By comparison, aap gene carriage was more common among susceptible S. epidermidis isolates (44%) than the MDR S. epidermidis isolates (27%). The atlE gene was the only gene that was found alone in the S. epidermidis genome. About 25% of the S. epidermidis isolates carried the atlE and aap genes and the ica operon simultaneously. In conclusion, rpoB gene amplicon sequencing is an easy and reliable method to identify CoNS isolates at the species and subspecies level. Both MDR and susceptible S. epidermidis isolates were found to be well equipped with adhesion and aggregation genes which might help them to adhere to artificial surfaces, colonize hospitalised patients and cause biofilm-related infections.

The activities of tigecycline and comparative agents on staphylococci and enterococci isolated from patients at general hospital wards (GHWs) and intensive care units (ICUs) at 3 university hospitals in Sweden were investigated. Oxacillin disc diffusion and minimal inhibitory concentration with E-test were used. The presence of mecA, vanA or vanB genes was determined with PCR. Statistically significant higher incidence of clindamycin, fusidic acid, rifampicin and multidrug-resistant CoNS was found at ICUs compared to GHWs. Resistance rates were low among S. aureus. Tigecycline, linezolid and vancomycin were the only agents with high activity against methicillin-resistant S. aureus and multidrug-resistant CoNS. Resistance rates were low among E. faecalis, except for high-level gentamicin-resistant (HLGR) E. faecalis. E. faecium showed high resistance rates to ampicillin, piperacillin/tazobactam and imipenem. The HLGR rates among E. faecium were lower than the rates for E. faecalis. Tigecycline and linezolid were the only drugs with high activity against all enterococci including vancomycin-resistant enterococci. No statistically significant differences in susceptibility rates were found between the ward levels for S. aureus and enterococcal isolates and no statistically significant differences were found between the hospitals.

BACKGROUND: The purpose of this study was to estimate the cost-effectiveness of using individual-donor nucleic acid testing (ID-NAT) in addition to serologic tests compared with the sole use of serologic tests for the identification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among blood donors in Sweden. STUDY DESIGN AND METHODS: The two strategies analyzed were serologic tests and ID-NAT plus serologic tests. A health-economic model was used to estimate the lifetime costs and effects. The effects were measured as infections avoided and quality-adjusted life-years (QALYs) gained. A societal perspective was used. RESULTS: The largest number of viral transmissions occurred with serologic testing only. However, the risks for viral transmissions were very low with both strategies. The total cost was mainly influenced by the cost of the test carried out. The cost of using ID-NAT plus serologic tests compared to serologic tests alone was estimated at Swedish Krona (SEK) 101 million (USD 12.7 million) per avoided viral transmission. The cost per QALY gained was SEK 22 million (USD 2.7 million). CONCLUSION: Using ID-NAT for testing against HBV, HCV, and HIV among blood donors leads to cost-effectiveness ratios that are far beyond what is usually considered cost-effective. The main reason for this is that with current methods, the risks for virus transmission are very low in Sweden.

Bacterial numbers in broth cultures were determined by bioluminescence assay of intracellular bacterial ATP. Broth MICs for strains of Staphylococcus epidermidis (ATCC 14990 and 35984) and Staphylococcus aureus (ATCC 25923, 29213 and 6538) were determined for cultures with different inocula (10⁵–10⁸ bacteria/ml) after 24 h of incubation in supplemented Mueller–Hinton broth containing vancomycin. All of the tested strains except one were susceptible to methicillin, and all of the strains were susceptible to vancomycin. Free vancomycin concentrations in the broth cultures of all strains were determined with an agar well bioassay after 24 h of incubation. Free vancomycin concentrations and bacterial numbers of ATCC 35984 and ATCC 29213 were also determined after 0.5, 2, 4, and 8 h. In a low inoculum (10⁵ bacteria/ml), the broth MICs were 1–4 μg/ml. In a high inoculum (∼10⁸ bacteria/ml), the broth MICs increased two- to fourfold to 4–8 μg/ml. In dense inocula (∼10⁹–10¹⁰ bacteria/ml), the concentrations of free vancomycin in the broth were reduced, in most cases below the detection limit of the bioassay (≤0.5 μg/ml). This reduction of free vancomycin was fast, occurring in initially dense inocula in less than 30 min. No emergence of resistance was seen. These results show a rapid reduction of free vancomycin in the broth and a simultaneous increase in broth MICs in high inocula, without development of resistance. This indicates that the dosing regimen of vancomycin is of particular importance in staphylococcal infections with dense inocula, e.g. infective endocarditis.

The objective of this study was to examine a design for a World Wide Web-based decision-support system in use by clinically active physicians. A prototype implementation of the design concerned management of infective endocarditis patient cases. The design was based on an integration of hypertext and rule-based knowledge. In the study sessions, physicians in the field of internal medicine worked on managing authentic patient cases in a laboratory setting. Data was collected from interviews with the physicians using video recordings and stimulated recall technique. The qualitative data was analysed according to the constant comparative method in order to develop a model of the physicians' usage of the system. The resulting model describes perceived contributions and criteria for usefulness of the system. The ways the physicians used the system showed that it was able to provide patient-specific support for confirming clinical decisions, for higher-level patient management, and for preparing for and initiating expert consultations. Users also stated that new medical knowledge could be gained as a side effect of using the system.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100 nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1β and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

The aim of the study was to describe the prevalence and age distribution of bacterial vaginosis (BV) during an observation period of 15 years in a population study with cross-sectional samples of adult women living on the Aland Islands. The Aland Islands form an archipelago in the Baltic Sea and are a province of Finland. Every fifth year, specific age groups in the adult female population are invited to participate in a screening program for early diagnosis of cervical cancer using a papanicolaou (PAP)-stained vaginal smear. Women in the age groups of 20, 25, 30, 35, 40, 45, 50, 55, and 60 years are called each year. BV diagnosis of the PAP-stained smears uses the classification according to Nugent. The PAP-stained smears from the screening program of cervical cancer 1993, 1998, 2003, and 2008 were used in this study. A total of 3456 slides were investigated and 271 women could be followed for the 15-year observation period. The prevalence of BV declined from 15.6% in 1993 to 8.6% in 2008. The highest prevalence occurred among the age groups of 35 and 50 years. Among the 271 women who could be followed for the 15-year observation period, two-third showed normal/intermediate flora and one-third were infected with BV at least once. As this is a cross-sectional population study spanning 15 years, the prevalence of BV in the female adult population of the Aland Islands can be estimated. The prevalence has declined between 1993 and 2008 from 15.6% to 8.6%.

Since bacterial vaginosis (BV) is characterized by a lack of, or very few, lactobacilli and high numbers of small, mostly anaerobic bacteria, an obvious treatment modality would be eradication of the BV-associated bacterial flora followed by reintroduction of lactobacilli vaginally. As probiotic treatment with lactobacilli is one tool for improving the cure rate when treating BV, it is necessary to know the length of time after treatment that clindamycin can be found in the vagina and if this could interfere with the growth of the probiotic lactobacilli. We evaluated the vaginal concentration of clindamycin in 12 women for 8 days to obtain data on the concentration of clindamycin in the vagina after intravaginal treatment with the drug. The participants were examined five times between two menstrual periods: before treatment, the day after treatment was finished, and 3, 5 and 8 days post-treatment. The first day post-treatment clindamycin 0.46 x 10-3 to 8.4 x 10-3 g/g vaginal fluid (median 2.87 x 10-3) was found. Thereafter, the concentration of clindamycin decreased rapidly. In 10 patients clindamycin was found after 3 days. A very low concentration was still present 5 days after treatment in four patients. After 8 days no clindamycin was found. Clindamycin is rapidly eliminated from the vagina, within 3-8 days, after local administration. Our results indicate that treatment with probiotic lactobacilli could be problematic if carried out within 5 days after cessation of clindamycin treatment.

Pseudomonas aeruginosa is 1 of the bacteria most adaptive to anti-bacterial treatment. Previous studies have shown nosocomial spread and transmission of clonal strains of P. aeruginosa in European hospitals. In this study we investigated antibiotic susceptibility and clonality in 101 P. aeruginosa isolates from 88 patients admitted to 8 Swedish ICUs during 2002. We also compared phenotypes and genotypes of P. aeruginosa and carried out cluster analysis to determine if phenotypic data can be used for surveillance of clonal spread. All isolates were collected on clinical indication as part of the NPRS II study in Sweden and were subjected to AFLP analysis for genotyping. 68 isolates with unique genotypes were found. Phenotyping was performed using MIC values for 5 anti-pseudomonal agents. Almost 6% of the isolates were multi-drug resistant (MDR), and this figure rose to almost 8% when intermediate isolates were also included. We found probable clonal spread in 9 cases, but none of them was found to be an MDR strain. Phenotypical cluster analysis produced 40 clusters. Comparing partitions did not demonstrate any significant concordance between the typing methods. The conclusion of our study is that cross-transmission and clonal spread of MDR P. aeruginosa does not present a clinical problem in Swedish ICUs, but probable cross-transmission of non-MDR clones indicate a need for improved hygiene routines bedside. The phenotype clusters were not concordant with genotype clusters, and genotyping is still recommended for epidemiological tracking.

Background: A study was designed to assess a computer-based program for continuous registration of antibiotic resistance, statistics concerning severity of illness, and consumption of antibacterial drugs.

Methods: The frequency of antibiotic resistance among bacteria in eight ICUs in southeastern Sweden was investigated yearly from 1995 through 1997. The antibiotic consumption in the ICUs was registered as defined daily doses (DDD) and compared to severity of illness (APACHE-II scores).

Results: There was a statistically significant increase in ampicillin resistance among Enterococcus spp. between 1996 and 1997, which was due to a shift from Enterococcus faecalis to Enterococcus faecium. A high prevalence of resistance among coagulase-negative staphylococci to oxacillin (≈ 70%), ciprofloxacin (≈ 50%), fucidic acid (≈ 50%) and netilmicin (≈ 30%) was seen in all ICUs during the whole study period. There was a statistically significant increase in ciprofloxacin resistance among Escherichia coli and Enterococcus spp. The resistance among Enterobacter spp. to cefotaxime decreased but this change was not statistically significant. Efforts were made to avoid betalactam antibiotics, except carbapenems, for treatment of infections caused by Enterobacter spp. and the consumption of cephalosporins decreased whereas the consumption of carbapenems increased. The total antibiotic consumption decreased by 2.5% during the study period. There was no correlation between APACHE II scores and antibiotic consumption.

Conclusions: Each ICU within a hospital ought to have a program for "on-line" antibiotic resistance surveillance of drugs used in that unit so that changes in empirical treatment can be made when there is an increase in antibiotic-resistant isolates within that unit.

Annual outbreaks of seasonal influenza are controlled or prevented through vaccination in many countries. The seasonal vaccines used are either inactivated, currently administered parenterally, or live-attenuated given intranasally. In this study three mucosal adjuvants were examined for the influence on the humoral (mucosal and systemic) and cellular influenza A-specific immune responses induced by a nasally administered vaccine. We investigated in detail how the anionic Endocine™ and the cationic adjuvants N3OA and N3OASq mixed with a split inactivated influenza vaccine induced influenza A-specific immune responses as compared to the vaccine alone after intranasal immunization. The study showed that nasal administration of a split virus vaccine together with Endocine™ or N3OA induced significantly higher humoral and cell-mediated immune responses than the non-adjuvanted vaccine. N3OASq only significantly increased the cell-mediated immune response. Furthermore, nasal administration of the influenza vaccine in combination with any of the adjuvants; Endocine™, N3OA or N3OASq, significantly enhanced the mucosal immunity against influenza HA protein. Thus the addition of these mucosal adjuvants leads to enhanced immunity in the most relevant tissues, the upper respiratory tract and the systemic circulation. Nasal influenza vaccination with an inactivated split vaccine can therefore provide an important mucosal immune response, which is often low or absent after traditional parenteral vaccination.

Purpose: Respiratory syncytial virus (RSV) is the most common cause of severe respiratory tract infection in infants. The aim was to identify host defence components in nasopharyngeal aspirate (NPA) from infants with RSV infection and to study the expression of the novel 25 kDa innate immunity protein SPLUNC1. less thanbrgreater than less thanbrgreater thanExperimental design: NPAs from infants were analyzed with 2-DE and MS in a pilot study. The levels of SPLUNC1 were analyzed with immunoblotting in 47 NPAs, admitted for RSV diagnosis. less thanbrgreater than less thanbrgreater thanResults: Totally, 35 proteins were identified in NPA, including several innate immunity proteins such as group X phospholipase A(2), different S100 proteins and SPLUNC1. In addition, a new truncated 15 kDa form of SPLUNC1 was identified that was detected in about 50% of the aspirates admitted for RSV diagnosis. RSV-positive boys had significantly less 25 kDa SPLUNC1 than RSV-negative boys while there were no significant differences among girls. less thanbrgreater than less thanbrgreater thanConclusions and clinical relevance: Several important innate immunity proteins were identified in NPA. Notably, a new truncated form of the newly suggested anti-bacterial protein SPLUNC1 was found. It is possible that a decrease in SPLUNC1 in the upper airways may increase the risk for severe pneumonia in boys.

[No abstract available]

Mobiluncus, a member of the anaerobic vaginal flora typical for bacterial vaginosis, is a strikingly mobile, curved, rod-shaped bacterium forming a bacterial genus that has thus far not been assigned with certainty to higher bacterial taxa. Two species, M curtisii and M mulieris, are well recognised, but atypical strains do occur. A normal residence for Mobluncus in the indigenous human bacterial flora might be the rectum or large intestine. Investigations during the last 20 years have not come up with a definite individual role for Mobiluncus in the pathogenesis of bacterial vaginosis. Its possible role in obstetric and gynaecological infections that are strongly suspected to be linked to bacterial vaginosis, and its role in extragenital infections, remain elusive. Mobiluncus is thus a conspicuous member of the mixed anaerobic flora that is so characteristic of bacterial vaginosis. The role of Mobiluncus in the pathogenesis of bacterial vaginosis will probably be resolved only when new insight is attained into the complex interrelationships of mixed anaerobic infections.

Diagnosing bacterial vaginosis (BV) has long been based on the clinical criteria of Amsel et al., whereby three of four defined criteria must be satisfied. Though there are other criteria and scoring methods which function well in comparison (i.e. Nugent scoring), it is not certain that they will always identify the same category of patients. Point-of-care methods based on various combinations of microbial products, presence of RNA, or more complex laboratory instrumentations such as sensor arrays, have also been introduced for the diagnosis of BV No method for diagnosing BV can at present be regarded as the best. It could be that - based partly on tacit knowledge on the part of the clinical investigators scoring in the clinic - various scoring systems have been chosen to fit a particular BV-related problem in a particular population. In this review we critically examine these pertinent issues influencing clinical scoring and laboratory diagnostics of BV. Copyright © APMIS 2005.

An international workshop on vaginal smear-based diagnosis of bacterial vaginosis was organized where 13 investigators scoring 258 slides with smears from vaginal fluid. Interobserver reproducibility of interpretations of Nugent scores, Hay/Ison scores and wet smear scores for the diagnosis of bacterial vaginosis was shown to be high. Detailed analysis of individual scoring results however indicated that basic standards of quality control to ensure robust individual readings of slides must be adhered to.

In a previous randomized controlled trial (LOGIP trial) the addition of local collagen-gentamicin reduced the incidence of postoperative sternal wound infections (SWI) compared with intravenous prophylaxis only. Consequently, the technique with local gentamicin was introduced in clinical routine at the two participating centers. The aim of the present study was to re-evaluate the technique regarding the prophylactic effect against SWI and to detect potential shifts in causative microbiological agents over time. All patients in this prospective two-center study received prophylaxis with application of two collagen-gentamicin sponges between the sternal halves in addition to routine intravenous antibiotics. All patients were followed for 60 days postoperatively. From January 2007 to May 2008, 1359 patients were included. The 60-day incidences of any SWI was 3.7% and of deep SWI 1.5% (1.0% mediastinitis). Both superficial and deep SWI were significantly reduced compared with the previous control group (OR=0.34 for deep SWI, Pless than0.001). There was no increase in the absolute incidence of aminoglycoside resistant agents. The majority of SWI were caused by coagulase-negative staphylococci (CoNS). The incidence of deep SWI caused by Staphylococcus aureus was 0.07%. The results indicate a maintained effect of the prophylaxis over time without absolute increase in aminoglycoside resistance.

otavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca(2+) concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP(3)), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT(3) receptor antagonists.

The Swedish Reference Group for Antibiotics (SRGA) has carried out a risk–benefit analysis of aminoglycoside treatment based on clinical efficacy, antibacterial spectrum, and synergistic effect with beta-lactam antibiotics, endotoxin release, toxicity, and side effects. In addition, SRGA has considered optimal dosage schedules and advice on serum concentration monitoring, with respect to variability in volume of drug distribution and renal clearance. SRGA recommends that aminoglycoside therapy should be considered in the following situations: (1) progressive severe sepsis and septic shock, in combination with broad-spectrum beta-lactam antibiotics, (2) sepsis without shock, in combination with broad-spectrum beta-lactam antibiotics if the infection is suspected to be caused by multi-resistant Gram-negative pathogens, (3) pyelonephritis, in combination with a beta-lactam or quinolone until culture and susceptibility results are obtained, or as monotherapy if a serious allergy to beta-lactam or quinolone antibiotics exists, (4) serious infections caused by multi-resistant Gram-negative bacteria when other alternatives are lacking, and (5) endocarditis caused by difficult-to-treat pathogens when monotherapy with beta-lactam antibiotics is not sufficient. Amikacin is generally more active against extended-spectrum beta-lactamase (ESBL)-producing and quinolone-resistant Escherichia coli than other aminoglycosides, making it a better option in cases of suspected infection caused by multidrug-resistant Enterobacteriaceae. Based on their resistance data, local drug committees should decide on the choice of first-line aminoglycoside. Unfortunately, aminoglycoside use is rarely followed up with audiometry, and in Sweden we currently have no systematic surveillance of adverse events after aminoglycoside treatment. We recommend routine assessment of adverse effects, including hearing loss and impairment of renal function, if possible at the start and after treatment with aminoglycosides, and that these data should be included in hospital patient safety surveillance and national quality registries.

Local infection control measures, antibiotic consumption and patient demographics from 1999-2000 together with bacteriological analyses were investigated in 29 ICUs participating in the ICU-STRAMA programme. The median antibiotic consumption per ICU was 1147 (range 605-2143) daily doses per 1000 occupied bed d (DDD₁₀₀₀). Antibiotics to which >90% of isolates of an organism were susceptible were defined as treatment alternatives (TA₉₀). The mean number of TA₉₀ was low (1-2 per organism) for Enterococcus faecium (vancomycin:VAN), coagulase negative staphylococci (VAN), Pseudomonas aeruginosa (ceftazidime:CTZ, netilmicin: NET) and Stenotrophomonas maltophilia (CTZ, trimethoprim-sulfamethoxazole: TSU), but higher (3-7) for Acinetobacter spp. (imipenem:IMI, NET, TSU), Enterococcus faecalis (ampicillin:AMP, IMI, VAN), Serratia spp. (ciprofloxacin:CIP, IMI, NET), Enterobacter spp. (CIP, IMI, NET, TSU), E. coli (cefuroxime:CXM, cefotaxime/ceftazidime:CTX/CTZ, CIP, IMI, NET, piperacillin-tazobactam:PTZ, TSU), Klebsiella spp. (CTX/CTZ CIP, IMI, NET, PTZ, TSU) and Staphylococcus aureus (clindamycin, fusidic acid, NET, oxacillin, rifampicin, VAN). Of S. aureus isolates 2% were MRSA. Facilities for alcohol hand disinfection at each bed were available in 96% of the ICUs. The numbers of TA₉₀ available were apparently higher than in ICUs in southern Europe and the US, despite a relatively high antibiotic consumption. This may be due to a moderate ecological impact of the used agents and the infection control routines in Swedish ICUs.

Data evaluation and classification have been made on measurements by an electronic nose on the headspace of samples of different types of bacteria growing on petri dishes. The chosen groups were: Escherichia coli, Enterococcus sp., Proteus mirabilis, Pseudomonas aeruginosa, and Staphylococcus saprophytica. An approximation of the response curve by time was made and the parameters in the curve fit were taken as important features of the data set. A classification tree was used to extract the most important features. These features were then used in an artificial neural network for classification. Using the ‘leave-one-out’ method for validating the model, a classification rate of 76% was obtained

It has been hypothesized that nosocomial enterococci might have virulence factors that enhance their ability to colonise hospitalised patients. The objectives of this study were to investigate the prevalence of genes encoding 3 virulence factors: aggregation substance (asa1), enterococcal surface protein (esp), and 5 genes within the cytolysin operon (cylA, cylB, cylM, cylL(L), cylL(S)) and cytolysin production in 115 enterococcal clinical isolates (21 Enterococcus faecium and 94 E. faecalis). Adhesion to siliconized latex urinary catheters in relation to presence of esp was analysed in a subset of isolates. The isolates were previously characterised by pulsed-field gel electrophoresis (PFGE). esp was the only virulence gene found in E. faecium. It was found in 71% of the 21 E. faecium isolates. asa1, esp, and the cyl operon were found in 79%, 73% and 13% respectively, of the 94 E. faecalis isolates. There was a complete agreement between presence of the cyl operon and phenotypic cytolysin production. Isolates belonging to a cluster of genetically related isolates carried esp and asa1 more often when compared to unique isolates. No difference was found with respect to cyl genes. E. faecalis isolates adhered with higher bacterial densities than E. faecium. E. faecalis isolates within the same PFGE cluster adhered with similar bacterial densities, but there was no association between adhesion and the presence of esp when isolates within the same cluster were compared. In conclusion, E. faecalis isolates with high-level gentamicin resistance (HLGR) belonging to clusters of genetically related isolates widely distributed in Swedish hospitals, were likely to carry both esp and asa1. Adhesion was not affected by esp.   

Leucocyte esterase (LE) in first-voided urine (FVU) and presence of leucocytes in urethral and cervical smears were evaluated to identify Mycoplasma genitalium infection in 416 men and 417 women attending Department of Genitourinary Medicine. M. genitalium was diagnosed in FVU specimens by realtime polymerase chain reaction. The prevalence of M. genitalium was 6.5% in women and 6.7% in men. In total, 88.5% (23/26) of M. genitalium-infected men were identified by a combination of urethral smear and the LE test. In women, the combination of urethral and/or cervical smears and/or a positive LE test identified 91.3% (21/23) of M. genitalium-infected patients. Organism load in FVU correlated significantly with presence of urethritis (> or =4 leucocytes per high-power field) in men. A combination of LE testing of urine and urethral and/or cervical smears can be used as screening tests to select patients for specific M. genitalium testing. By this strategy, about 10% of infected individuals will remain undetected.

Chlamydia pneumoniae has during recent years been associated with cardiovascular disease and atherosclerosis. Chemokines, leukocyte adhesion proteins and metalloproteinases are significant for chemotaxis and attachment of leukocytes to vessel walls, and for stability of atherosclerotic plaques. To determine the ability of C. pneumoniae to elicit inflammation in a relevant target host cell, we infected human coronary artery endothelial cells (HCAEC) with a clinical isolate of C. pneumoniae. Extracellular release of five chemokines, two adhesion proteins and a metalloproteinase was measured at different time points after infection using a cytometric bead assay and ELISA. Secretion of IL-8, MCP-1, MIG, IP-10 and ICAM-1 was significantly increased 48 h after C. pneumoniae infection of HCAEC in comparison with uninfected controls. Release of RANTES occurred already 6 h after infection. C. pneumoniae did not elicit release of E-selectin or MMP-1. We conclude that C. pneumoniae induces expression of proinflammatory components in HCAEC, which would promote migration of leukocytes towards endothelial cells. This suggests that C. pneumoniae initiates and propagates vascular inflammation in ways that contribute to coronary artery disease.

Background: Nitric oxide (NO) is produced in macrophages by the inducible NO synthase (iNOS) upon activation by pro-inflammatory cytokines. NO has been shown to be essential for the control of Mycobacterium tuberculosis infection in murine models whereas its importance in man is not as clear. There is a lack of studies regarding the susceptibility to reactive nitrogen species (RNS) in clinical strains of M. tuberculosis and the relation to first-line drug resistance, such as to isoniazid (INH). The aim of this study was to explore susceptibility to RNS and intracellular survival of clinical strains of M. tuberculosis, with or without INH resistance.

Method: Seven clinical strains of M. tuberculosis were transformed with the pSMT1-plasmid encoding Vibrio harveyi luciferase. Survival was analysed by luminometry following exposure to the NO donor DETA/NO or peroxynitrite (SIN-1). Intracellular killing was studied in murine macrophages (RAW 264.7) activated with interferon gamma (IFN-γ) and lipopolysaccharide (LPS).

Results: There was a significant effect on growth control of M. tuberculosis strains upon macrophage activation, which showed variability among clinical isolates. In the cell-free system, all strains showed a dose-dependent susceptibility to DETA/NO and SIN-1, and clinical strains were in general more resistant than H37Rv to DETA/NO. INH-resistant strains with an inhA mutation were significantly more tolerant to DETA/NO than inhA wild type.

Conclusion: Reactive nitrogen species inhibited growth of clinical M. tuberculosis isolates both in an intra- and extracellular model with significant difference between strains. Increased tolerance to NO was associated with isoniazid resistance mediated by inhA.

Background: Signature matching of nucleotide sequences in the V1 and V3 regions 16S rRNA genes using pyrosequencing technology is a powerful tool for typing vaginal Lactobacilli to the species level and has been used for investigating the vaginal microbial niche. Methods: This study has characterized the normal cultivable vaginal Lactobacillus flora at varying estradiol levels in plasma, the study comprised 17 patients undergoing ovarian stimulation for In Vitro Fertilization (IVF) treatment. The vaginal status of each participant was initially assessed as normal according to Amsel and Nugent criteria. Results: L. crispatus, L. gasseri and/or L. jensenii were present in 10of the patients throughout the study period, and little variation among these three species was encountered in individual patients. The flora of three women was dominated by L. delbrüeckii, L. rhamnosus or L. vaginalis. One woman exhibited a dominance of L. iners. The flora of the remaining three women were initially dominated by L. rhamnosus or L. reuteri, but as their estrogen levels rose, their flora composition altered, to become dominated by one of the three species most common in a normal, healthy vagina. Conclusion: Signature matching of nucleotide sequences in the V1 and V3 regions of 16S rRNA genes is a discriminative tool for the study of vaginal Lactobacilli and can be used to track the Lactobacillus flora under a variety of physiological conditions. © 2008 Jakobsson and Forsum, licensee BioMed Central Ltd.

Background: Signature matching of nucleotide sequences in the V1 and V3 regions 16S rRNA genes using pyrosequencing technology is a powerful tool for typing vaginal Lactobacilli to the species level and has been used for investigating the vaginal microbial niche.

Methods: This study has characterized the normal cultivable vaginal Lactobacillus flora at varying estradiol levels in plasma; the study comprised 17 patients undergoing ovarian stimulation for In Vitro Fertilization (IVF) treatment. The vaginal status of each participant was initially assessed as normal according to Amsel and Nugent criteria.

Results: L. crispatus, L. gasseri and/or L. jensenii were present in 10 of the patients throughout the study period, and little variation among these three species was encountered in individual patients. The flora of three women was dominated by L. delbrüeckii, L. rhamnosus or L. vaginalis. One woman exhibited a dominance of L. iners. The flora of the remaining three women were initially dominated by L. rhamnosus or L. reuteri, but as their estrogen levels rose, their flora composition altered, to become dominated by one of the three species most common in a normal, healthy vagina.

Conclusion: Signature matching of nucleotide sequences in the V1 and V3 regions of 16S rRNA genes is a discriminative tool for the study of vaginal Lactobacilli and can be used to track the Lactobacillus flora under a variety of physiological conditions.

[No abstract available]

Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described, however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A subsequent database search revealed that the amino acid sequence of a lysine-rich C-terminal segment of HP0508 is identical to the C terminus of HP0863. Recombinant proteins expressed from HP0508 and HP0863 bound plasminogen specifically and in a lysine-dependent manner. We designate these genes pgbA and pgbB, respectively. These proteins are expressed by a variety of H. pylori strains, have surface-exposed domains, and do not inhibit plasminogen activation. These results indicate that pgbA and pgbB may allow H. pylori to coat its exterior with plasminogen, which subsequently can be activated to plasmin. The surface acquisition of protease activity may enhance the virulence of H. pylori.

Aichi virus (AiV), genus Kobuvirus, family Picornaviridae, is associated with gastroenteritis in humans. Previous studies have shown high seroprevalence but low incidence (0.9-4.1%) in clinical samples. We report here the first detection of AiV in Sweden. Two hundred twenty-one specimens from hospitalized patients with diarrhea, who were negative for other enteric viruses, were included in the study. AiV were detected in three specimens, all from elderly patients. Phylogenetic analysis revealed that the three Swedish isolates belonged to genotype A and were genetically closest to European and Asian strains of AiV.

UNLABELLED: ABSTRACT:

BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.

RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.

CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.

The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.