Effects of selective decontamination of digestive tract on mortality and acquisition of resistant bacteria in intensive care: a randomised controlled trial.

BACKGROUND: Selective decontamination of the digestive tract (SDD) is an infection-prevention regimen used in critically ill patients. We assessed the effects of SDD on intensive-care-unit (ICU) and hospital mortality, and on the acquisition of resistant bacteria in adult patients admitted to intensive care. METHODS: We did a prospective, controlled, randomised, unblinded clinical trial. 934 patients admitted to a surgical and medical ICU were randomly assigned oral and enteral polymyxin E, tobramycin, and amphotericin B combined with an initial 4-day course of intravenous cefotaxime (SDD group n=466), or standard treatment (controls n=468). Primary endpoints were ICU and hospital mortality and the acquisition of resistant bacteria. FINDINGS: In the SDD group 69 (15%) patients died in the ICU compared with 107 (23%) in the control group (p=0.002). Hospital mortality was lower in the SDD groups than in the control group (113 [24%] vs 146 [31%], p=0.02). During their stay in intensive care, colonisation with gram-negative bacteria resistant to ceftazidime, ciprofloxacin, imipenem, polymyxin E, or tobramycin occurred in 61 (16%) of 378 SDD patients and in 104 (26%) of 395 patients in the control group (p=0.001). Colonisation with vancomycin-resistant enterococcus occurred in five (1%) SDD patients and in four (1%) controls (p=1.0). No patient in either group was colonised with meticillin-resistant Staphylococcus aureus. INTERPRETATION: In a setting with low prevalence of vancomycin-resistant enterococcus and meticillin-resistant S aureus, SDD can decrease ICU and hospital mortality and colonisation with resistant gram-negative aerobic bacteria.

A boy with nosocomial malaria tropica contracted in a Dutch hospital.

A 12-year-old Dutch boy was admitted because of severe neurotrauma after a traffic accident. On day 33 after admission, a Plasmodium falciparum infection was found in a routine blood smear. Most probably he was infected by blood of a patient next to him, a girl with severe malaria tropica. The genotype of the P. falciparum in both patients was identical.

Effect of albumin on the photodynamic inactivation of microorganisms by a cationic porphyrin.

BACKGROUND: Photodynamic inactivation (PDI) employs visible light and a photosensitizer to inactivate cells. The technique is currently clinically used for the treatment of several malignancies. However, the PDI of microorganisms still remains in the research phase. PURPOSE: To study the effect of human blood plasma and human serum albumin (HSA) on the PDI of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. METHODS: PDI experiments were performed using white light (30 mW cm-2) and the cationic 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) as photosensitizer. RESULTS: The microorganisms could be successfully photoinactivated by TriP[4] when suspended in phosphate buffered saline (PBS). In this medium, P. aeruginosa was the most resistant microorganism. Changing the suspending medium from PBS to human blood plasma reduced the PDI of all three microorganisms. In human blood plasma C. albicans was the most resistant microorganism. The same results were obtained with 4.5% and 7% HSA/PBS suspensions. CONCLUSIONS: Albumin inhibits the PDI of S. aureus, P. aeruginosa and C. albicans in a dose dependent manner. However, our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans.

Group a streptococcal meningitis in adults: report of 41 cases and a review of the literature.

Chart review of 41 adult patients with group A streptococcal (GAS) meningitis in The Netherlands revealed that this is a community-acquired disease and occurs mainly in patients with predisposing factors: of the 41 patients in this case series, 24 (60%) of 40 evaluable patients had otitis or sinusitis. Fever and neck stiffness were the most common clinical manifestations of disease, but, in addition, high rates of seizures (12 [32%] of 38 patients), focal neurological findings (13 [36%] of 36 patients), and hyponatremia (20 [58%] of 35 patients) were found. In contrast with data from the literature that describes 27 adult cases, we found that GAS meningitis is a fulminant disease with a mortality rate of 27% (10 of 37 patients), and that neurological sequelae occur in 36% (12 of 38) of surviving patients.

Cytokine concentrations in seminal plasma from subfertile men are not indicative of the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract.

The inflammatory response to the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract of subfertile men without any signs or symptoms of infection was investigated by measuring the concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in seminal plasma. Semen samples were collected from 30 culture-positive subfertile males and 23 culture-negative subfertile males. Enzyme-linked immunosorbent assays showed that IL-8 was present in relatively high concentrations (0.12-4.8 ng/ml) in all semen samples investigated. In contrast, the other cytokines were only detectable in 72% (IFN-gamma), 44% (IL-6) and 19% (TNF-gamma) of the samples and were present in relatively low concentrations (1-410 pg/ml). Seminal plasma cytokine concentrations were similar in samples from culture-positive and culture-negative males. These data strongly indicate that the presence of U. urealyticum or M. hominis in the lower genital tract of subfertile males reflects a silent colonisation rather than infection.

Demonstration by a nested PCR for Mycoplasma pneumoniae that M. pneumoniae load in the throat is higher in patients hospitalised for M. pneumoniae infection than in non-hospitalised subjects.

A nested PCR protocol to detect Mycoplasma pneumoniae DNA in throat specimens was developed. An amplification control (AC) template, which is amplified by the same primers as the M. pneumoniae target sequence, was constructed. The assay allowed highly specific and sensitive detection of M. pneumoniae DNA. In all, 305 throat samples, 62 from hospitalised patients and 243 from non-hospitalised subjects, were analysed by the nested PCR. Inhibition of the PCR was observed in 20% of the samples, but was abolished after a 1 in 10 dilution. Throat samples from 5 (8%) of the hospitalised patients and from 7 (3%) of the non-hospitalised subjects were positive for M. pneumoniae DNA. To investigate the relationship between M. pneumoniae load and the severity of disease, the M. pneumoniae load in 10 throat samples from M. pneumoniae-positive subjects was assessed semi-quantitatively by application of the nested PCR to a series of limiting dilutions of nucleic acid extracted from these throat samples. The calculated M. pneumoniae load varied from 20 to 3830 cfu/ml of throat sample. The mean M. pneumoniae load in samples from the hospitalised patients was significantly higher than that in samples from the non-hospitalised subjects. The nested PCR is a useful tool to detect M. pneumoniae DNA in the throat and to study the relationship between the load of M. pneumoniae in throat samples and severity of disease due to M. pneumoniae infection.

Influence of infection of cells with bacteria associated with reactive arthritis on the peptide repertoire presented by HLA-B27.

Reactive arthritis (ReA) after infections with various gram-negative bacteria is strongly associated with the MHC class I molecule HLA-B27. It is supposed that the B27 molecule itself plays a role in the pathogenesis of ReA by presenting antigenic peptides to cytotoxic T lymphocytes. The peptide repertoires presented by Salmonella-, Shigella- and non-infected cells were compared to identify such peptides. From the peptides isolated from the B27 molecules of these cells, profiles were generated by reversed-phase chromatography and peaks present in the profiles from infected cells but not in profiles from non-infected cells were studied for their peptide compositions. Some sequences with identity to those in human histone H3, human ribosomal protein S17 and the heavy chain of HLA-B27 itself were detected only in profiles from infected cells. All peptides identified from infected cells contained the B*2705 peptide-binding motif. The data suggest that HLA-B27-positive cells infected with ReA-inducing bacteria show an increased presentation of certain self-peptides. There was no evidence for altered peptide-binding specificity of B27 after infection. However, the interpretations were hampered by the variation in peptide presentation between different experiments.

Assessment of Chlamydia trachomatis infection of semen specimens by ligase chain reaction.

Diagnostic potential of the Chlamydia trachomatis ligase chain reaction system (LCx) to assess the presence of C. trachomatis in urine and semen specimens was evaluated. Paired urine and semen specimens from 153 asymptomatic male partners of subfertile couples attending our Center for Reproductive Medicine were examined by LCx. As controls, 19 semen samples from four donors who were participating in the programme for artificial insemination were used. Of these, 12 samples had previously been shown to be C. trachomatis-positive by an in-house PCR. C. trachomatis was detected by LCx in seven of 153 (5 %) urine samples. None of the 153 semen samples tested positive by LCx. Also, none of the 12 C. trachomatis-containing control semen samples were positive by LCx. By in-house PCR, seven urine specimens and two of 153 (1 %) semen samples tested positive. The corresponding urine samples of these male partners were also C. trachomatis-positive, as well as the 12 C. trachomatis-containing samples from donors. In conclusion, LCx is not sensitive enough to assess the presence of C. trachomatis in semen specimens; therefore, this method is not recommended to routinely screen semen specimens from donors who participate in programmes for artificial insemination or male partners of subfertile couples for C. trachomatis.

Induced Helicobacter pylori vacuolating cytotoxin VacA expression after initial colonisation of human gastric epithelial cells.

To study the effect of initial colonisation on Helicobacter pylori gene expression, altered H. pylori gene transcription during co-culture with human gastric epithelial cells was determined. Therefore, an insertion library of H. pylori with random chromosomal fusions to a promoterless cat gene was grown in the presence of HM02 gastric epithelial cells and varying levels of chloramphenicol. One H. pylori transformant was chloramphenicol-resistant in the presence, but chloramphenicol-susceptible in the absence of gastric epithelial HM02 cells. This transformant had the promoterless cat gene inserted into the HP0887 gene, which encodes the vacuolating cytotoxin VacA, an important virulence factor of H. pylori. Reverse transcriptase polymerase chain reaction on cDNA of this transformant confirmed vacA upregulation near HM02 cells. These results show the applicability of this technique to study H. pylori gene regulation in its natural environment.

5-fluorocytosine-related bone-marrow depression and conversion to fluorouracil: a pilot study.

The aim of this study is to investigate whether fluorouracil (5-FU) could be responsible for bone-marrow depression occurring in fluorocytosine (5-FC) treated patients. Six 5-FC treated patients were included in this pilot study. Toxicity was monitored by means of thrombocyte and leucocyte counts. 5-FC and 5-FU serum levels were measured using a high-performance liquid chromatography (HPLC) assay that allows simultaneous determination of both compounds. The amounts of 5-FU in the 34 available serum samples remained below the limit of quantitation (< 0.05 mg/L), whereas 5-FC levels could be detected in all samples. Instead, low levels of the 5-FU catabolite alpha-fluoro-beta-alanine (FBAL) were detected in several of the investigated serum samples. In case of three patients thrombocyte counts remained within the normal range during 5-FC treatment, whereas one patient developed thrombocytopenia (50 x 10(9) thrombocytes/L) during therapy. Furthermore, one patient developed leucocytopenia (2.6 x 10(9) leucocytes/L) during 5-FC therapy, whereas the remaining five patients were suffering from leucocytosis prior to 5-FC therapy. In conclusion, we found nondetectable 5-FU serum concentrations (< 0.05 mg/L) in ICU patients treated with intravenous 5-FC, making it unlikely that 5-FC-associated toxicity results from 5-FU exposure in patients receiving intravenous 5-FC therapy. These findings may be explained by the fact that our patients received 5-FC intravenously instead of orally, therefore not allowing active conversion of 5-FC to 5-FU by the human intestinal microflora.

Peri-implant tissue is an important niche for Staphylococcus epidermidis in experimental biomaterial-associated infection in mice.

Biomaterial-associated infections (BAI), which are predominantly caused by Staphylococcus epidermidis, are a significant problem in modern medicine. Biofilm formation is considered the pivotal element in the pathogenesis, but in previous mouse studies we retrieved S. epidermidis from peri-implant tissue. To assess the kinetics and generality of tissue colonization, we investigated BAI using two S. epidermidis strains, two biomaterials, and two mouse strains. With small inocula all implants were culture negative, whereas surrounding tissues were positive. When higher doses were used, tissues were culture positive more often than implants, with higher numbers of CFU. This was true for the different biomaterials tested, for both S. epidermidis strains, at different times, and for both mouse strains. S. epidermidis colocalized with host cells at a distance that was >10 cell layers from the biomaterial-tissue interface. We concluded that in mouse experimental BAI S. epidermidis peri-implant tissue colonization is more important than biofilm formation.

Interleukin 1alpha increases the susceptibility of rabbits to experimental viridans streptococcal endocarditis.

Major predisposing conditions for infective endocarditis (IE) are the presence of a cardiac platelet-fibrin vegetation and of circulating bacteria with relatively low susceptibility to microbicidal activity of blood platelets. The influence of proinflammatory conditions on development of IE is unknown. We studied the effects of the presence of a catheter, inserted to induce platelet-fibrin vegetations, and of the proinflammatory cytokine interleukin-1alpha in rabbit experimental IE. Leaving the catheter in place after challenge with viridans streptococci predisposed for experimental IE. IE susceptibility rapidly decreased between 0 to 6 h after catheter removal. The catheter did not predispose for IE by providing a site for bacterial adherence, as almost all explanted catheters were culture negative. To mimic the proinflammatory influence of the catheter, rabbits were injected with interleukin-1alpha at 24 h after catheter removal and at 0, 1, and 3 h before bacterial challenge. Interleukin-1alpha injected 3 h prior to challenge significantly increased IE incidence due to a platelet releasate-susceptible Streptococcus oralis strain, with rapidly increasing numbers of bacteria within the vegetations. IE due to the Streptococcus sanguis strain less susceptible to platelet releasate was not enhanced. We conclude that proinflammatory stimuli, either a catheter or interleukin-1alpha, enhanced susceptibility to IE due to the platelet releasate-susceptible S. oralis. As with rabbits, temporary intravascular proinflammatory conditions may predispose for IE in humans at risk for this serious infection.

Underreporting of meningococcal disease incidence in the Netherlands: results from a capture-recapture analysis based on three registration sources with correction for false positive diagnoses.

In order to come to a reliable evaluation of the effectiveness of the chosen vaccination policy regarding meningococcal disease, the completeness of registrations on meningococcal disease in the Netherlands was estimated with the capture-recapture method. Data over 1993-1998 were collected from (A) mandatory notifications (n = 2926); (B) hospital registration (n = 3968); (C) laboratory surveillance (n = 3484). As the standard capture-recapture method does not take into account false positive diagnoses, we developed a model to adjust for the lack of specificity of our sources. We estimated that 1363 cases were not registered in any of the three sources in the period of study. The completeness of the three sources was therefore estimated at 49% for source A, 67% for source B and 58% for source C. After adjustment for false positive diagnoses, the completeness of source A, B, and C was estimated as 52%, 70% and 62%, respectively. The capture-recapture methods offer an attractive approach to estimate the completeness of surveillance sources and hence contribute to a more accurate estimate of the disease burden under study. However, the method does not account for higher-order interactions or presence of false positive diagnoses. Being aware of these limitations, the capture-recapture method still elucidates the (in)completeness of sources and gives a rough estimate of this (in)completeness. This makes a more accurate monitoring of disease incidence possible and hence attributes to a more reliable foundation for the design and evaluation of health interventions such as vaccination programs.

Photodynamic inactivation of fibroblasts by a cationic porphyrin.

An important determinant of the clinical applicability and value of antimicrobial photodynamic inactivation (PDI) is the cytotoxicity of the treatment to human cells. We evaluated the in vitro cytotoxicity of PDI to human dermal fibroblasts using 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) as the photosensitiser. The fibroblasts were exposed to a PDI regime that is known to be sufficient for the inactivation of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The PDI experiments were carried out in phosphate-buffered saline (PBS) and in 6.25%, 12.5%, 25% and 50% fetal calf serum (FCS)/PBS suspensions. Cell viability subsequent to exposure was evaluated after 0 h, 6 h and 18 h using the methylthiazoletetrazolium (MTT) assay and compared to pretreatment values. At a TriP[4] concentration previously demonstrated to induce a 5 log(10)-unit reduction in a viable count for S. aureus, 79% of the fibroblasts were photo-inactivated. Increasing the FCS concentration in the medium protected the fibroblasts against PDI. Based on our in vitro results, we propose that in vivo PDI of S. aureus holds potential; however, PDI of P. aeruginosa and C. albicans will probably require such a strong PDI regime that it will induce substantial damage to fibroblasts.

Human microsporidiosis: Clinical, diagnostic and therapeutic aspects of an increasing infection.

Human microsporidiosis is a parasitic infection due to species of four different genera: Encephalitozoon; Enterocytozoon; Nosema; and Pleistophora. Although well known as a cause of disease in animals, microsporidiosis was only occasionally reported in humans. Recently, in human immunodeficiency virus (HIV)-infected patients, microsporidia belonging to Encephalitozoon and Enterocytozoon species have proved to be important opportunistic pathogens. Enterocytozoon bieneusi is associated with chronic intermittent diarrhea, cholangiopathy and sinusitis whereas Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi, the three Encephalitozoon species found in humans, are associated with diarrhea, rhinosinusitis, keratoconjunctivitis, nephritis and hepatitis. Diagnosis of microsporidial infections in humans was until recently an invasive, laborious procedure including electron microscopy of small intestine biopsies. However, new simple staining methods using Uvitex 2B or modified trichrome stain for feces and other body fluids have facilitated clinical diagnosis as well as drug evaluation and epidemiological studies. The application of monoclonal antibodies and molecular techniques such as the polymerase chain reaction have further improved microsporidial diagnosis. Treatment of Entero. bieneusi has, until now, been unsuccessful whereas albendazole has proved to be an effective treatment for Encephalitozoon species infection. Identification of effective treatment for Entero. bieneusi infections and further study of the pathogenicity of these microsporidial infections in immunocompetent hosts are important future challenges.

Genes of non-typeable Haemophilus influenzae expressed during interaction with human epithelial cell lines.

Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.

Space-time cluster analysis of invasive meningococcal disease.

Clusters are recognized when meningococcal cases of the same phenotypic strain (markers: serogroup, serotype, and subtype) occur in spatial and temporal proximity. The incidence of such clusters was compared to the incidence that would be expected by chance by using space-time nearest-neighbor analysis of 4,887 confirmed invasive meningococcal cases identified in the 9-year surveillance period 1993-2001 in the Netherlands. Clustering beyond chance only occurred among the closest neighboring cases (comparable to secondary cases) and was small (3.1%, 95% confidence interval 2.1%-4.1%).

Characteristics of pathogenic Neisseria meningitidis in Moscow: prevalence of 'non-European' strains.

OBJECTIVE: To investigate the distribution of serogroups, serotypes and subtypes, and susceptibility to antibiotics, of 75 strains isolated from patients with systemic meningococcal disease in Moscow in 1993--95. RESULTS: In contrast to the situation in most European countries, 21% of group A strains were found. Sixty-nine per cent of the strains were non-serotypeable using the current panel of antibodies, and 21% of strains were non-subtypeable. Twenty-nine different serotype---subtype combinations were found among 69 strains of group A, B and C. No combination predominated clearly; relatively more frequent strains had the formulae B:NT:P1.2, A:4:P1.5, 10 and C:4:P1.10. Recently, such strains have been very rare in western Europe; in contrast, the strains predominating in western Europe were not found in Moscow. All strains were sensitive to penicillin, chloramphenicol and rifampicin. CONCLUSIONS: Moscow strains of Neisseria meningitidis demonstrated a substantial diversity of serotypes and subtypes that probably corresponded to a post-epidemic situation in Russia. The obvious difference in circulating strains and presumably in immunity of populations in western Europe and Russia increases the probability of mutual exchange of pathogenic strains and stresses the need for group B vaccine protecting from both western and eastern European variants of meningococci.

Antibiotic guidelines and antibiotic use in adult bacterial meningitis in The Netherlands.

In The Netherlands, national guidelines for the treatment of adult patients with bacterial meningitis were introduced in October 1997. In 1998 we began a prospective, nationwide study to evaluate the compliance with these consensus-based guidelines. In addition, we evaluated whether the recommended initial treatment provides adequate microbiological coverage. From October 1998 to January 2000, 365 adults with bacterial meningitis were identified using information from The Netherlands Reference Laboratory for Bacterial Meningitis; 263 patients were classified into four categories depending on patient's age and underlying health status. In the first category, patients 16-60 years without risk factors, Neisseria meningitidis was the most common pathogen (53%); 62 of 127 patients (49%) received treatment in compliance with the guidelines. In the second and third categories, patients >60 years without risk factors and those with risk factors independently of age, Streptococcus pneumoniae caused 61% and 58% of cases, respectively. Compliance in these categories was about 17%. Overall, 33% of patients received treatment in compliance with the guidelines. The microbiological coverage of patients treated in compliance and not in compliance with the guidelines was 98% and 93%, respectively. In conclusion, 1 year after national consensus-based guidelines for the initial treatment of adult patient with bacterial meningitis were introduced in The Netherlands, only one-third of Dutch physicians were adhering to the guidelines. The microbiological coverage for the patients who were treated in compliance with the guidelines was almost complete (98%).

An in vitro study on the active conversion of flucytosine to fluorouracil by microorganisms in the human intestinal microflora.

BACKGROUND: Investigation of the rate of active conversion of flucytosine to fluorouracil by microorganisms in the intestinal microflora. METHODS: Active conversion of flucytosine was investigated using viable and nonviable Escherichia coli at different flucytosine concentrations. Additionally, flucytosine conversion was studied in fecal specimens from 3 neutropenic patients at the start of the antimicrobial/antifungal prophylaxis (C/A regimen) and 1 week later. RESULTS: Flucytosine levels decreased by an average of 72, 71 and 72% flucytosine after incubation for 48 h of 10(10) viable E. coli /ml suspension in broth containing 13, 130 and 1300 mg/l flucytosine, respectively. The decreasing flucytosine levels corresponded approximately to an identical increase in fluorouracil levels. Also, a 44% decrease of flucytosine levels occurred when nonviable E. coli were used, indicating that bacterial viability is not necessary for this conversion. When fecal specimens of 2 patients were investigated prior to the C/A regimen, significant flucytosine conversion occurred, whereas this conversion was not observed in the corresponding fecal specimens after 1 week of C/A regimen. CONCLUSION: These in vitro experiments showed that extensive flucytosine conversion can occur in the human intestinal microflora by E. coli. Consequently, fluorouracil exposure and fluorouracil-related toxicity may occur in the flucytosine-treated patient.