Control of microbial contamination in dental unit water systems using tetra-sodium EDTA.

AIM: To examine the efficacy of tetra-sodium EDTA in controlling microbial contamination of dental unit water systems (DUWS). METHODS AND RESULTS: Ten dental units were treated once a week with either 4% or 8% tetra-sodium EDTA for four or two consecutive weeks, respectively. Before treatment, 43% and 60% of the water samples from the air/water triple syringe and high-speed hand-pieces, respectively, exceeded the American Dental Association (ADA) guidelines of 200 CFU ml(-1) water during a 6-week baseline period. After each weekend treatment, the levels of microbial contamination in all DUWS fell significantly (P < 0.001) to below the ADA guideline. By the end of the week, microbial counts in the outflowing water had returned to baseline levels indicating a transient effect of single doses of tetra-sodium EDTA, and the need for multiple applications. The biofilms were virtually eliminated after a single weekend treatment. CONCLUSIONS: Tetra-sodium EDTA is effective in controlling microbial contamination in DUWS. SIGNIFICANCE AND IMPACT OF THE STUDY: Inexpensive, effective and safe products for reducing the microbial load of water from DUWS are needed to meet ADA and other national guidelines. Tetra-sodium EDTA can significantly reduce microbial biofilms and bacterial counts in outflowing water, and is compatible for use in DUWS.

Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA.

AIMS: We examined the efficacy of tetrasodium EDTA in eradicating biofilms derived from salivary inocula or pure cultures of Candida albicans on discs of polymethyl methacrylate (PMMA) denture base or on toothbrushes that had been used normally for 4-8 weeks. Its efficiency in virus neutralization was also determined. METHODS AND RESULTS: Overnight (16 h) treatment with 4% (w/v) tetrasodium EDTA solution reduced salivary and C. albicans biofilm viable counts by > or =99%. Biofilm removal was confirmed using confocal laser scanning microscopy. Presence/absence of sucrose during biofilm formation had no effect on killing efficacy. Prolonged treatment of PMMA with tetrasodium EDTA did not influence subsequent formation of C. albicans biofilms or affect surface roughness of the PMMA, but it reduced subsequent biofilm formation from a salivary inoculum. Infectivities of herpes simplex virus and polio virus suspensions were reduced by >99.99% by treatment for 1 and 2 h, respectively. CONCLUSIONS: Tetrasodium EDTA solution efficiently disinfected toothbrushes and PMMA discs, with the detachment of biofilms, and rapidly neutralized both nonenveloped and enveloped viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Dentures and toothbrushes become contaminated by bacterial biofilms and by viruses. There is a need for disinfection methods that are rapidly effective, cost-effective, nontoxic and easily implemented. These studies indicate that tetrasodium EDTA solution has disinfection applications in the oral care field.

Intravascular catheters and biofilm control.

Intravascular catheters are the most common cause of nosocomially acquired bloodstream infections. Bacteria found adhering to the intraluminal surfaces of catheters are the principal source and cause of these infections. Adherent bacteria overtime are known to form multicellular communities which become encased within a three dimensional matrix of extracellular polymeric material known as biofilms, which are thought to be responsible for persistent infections. Consequently, a number of technologies have been developed to help prevent and control biofilms in intravascular catheters. One such approach involves impregnating catheter material with antimicrobial agents. Unfortunately these methods are not universally effective in preventing catheter-related biofilm infections. Technologies that utilise antimicrobials, as catheter locks have been shown to have more potential for preventing biofilm formation and reducing the incidences of catheter related bloodstream infections (CRBSI). This article discusses the significance of biofilms in intravascular catheters and determines whether the treatments available today are proving to be effective for controlling biofilms and draws attention to future avenues which are being investigated to control biofilms and therefore CRBSI.

In situ endoluminal brushing: a safe technique for the diagnosis of catheter-related bloodstream infection.

We assessed the safety of the endoluminal brush technique for the in situ diagnosis of central-venous-catheter (CVC)-related bloodstream infection (CRBSI). The endoluminal brush was used to investigate patients with suspected CRBSI by sampling the CVC lumen to within 3-5 cm of the catheter tip (the brush was kinked to mark the length required). Quantitative peripheral blood cultures were taken 1 min pre-brushing and 1 min and 1h post-brushing. In addition, a 1 mL sample of through-catheter blood was aspirated immediately pre- and post-brushing, and aliquots were directly plated on to blood agar to determine the load of CVC-associated bacteria. After CVC removal, catheter tips were sent for culture using the Maki roll and modified Cleri flush techniques. Of 139 suspected cases, 61 (45%) had confirmed episodes of CRBSI. No patients experienced fever or rigors, or had evidence of embolic phenomena after CVC endoluminal brushing. There was no difference overall in pre-brushing and 1-min post-brushing peripheral blood bacterial counts (P < 0.702). Peripheral and CVC blood bacterial counts were significantly reduced 1h post-brushing (P < 0.03 and P < 0.001, respectively), possibly due to removal of endoluminal biomass. The endoluminal brush technique can be safely used to diagnose CRBSI without the need for CVC removal, provided that the brush is not allowed to protrude beyond the tip of the catheter.

Use of in vivo-generated biofilms from hemodialysis catheters to test the efficacy of a novel antimicrobial catheter lock for biofilm eradication in vitro.

This biofilm study was conducted to assess the in vitro activity of tetrasodium EDTA on catheters that had been routinely removed from hemodialysis patients at Leeds Teaching Hospitals Trust due to maturation of fistula. Catheters were screened by culture of through-catheter flush, and isolates were identified by standard methodologies; 20 isolates were found to be biofilm positive. Initial biofilm cell count levels averaged above 10(5) CFU/1-cm catheter section. Bacteria identified in the biofilms were gram-negative (1 isolate), gram-positive (11 isolates), or mixed species (8 isolates). After a 24-h lock, 40 mg of tetrasodium EDTA per ml was effective at eradicating the total biofilm viable count in almost all cases. The efficacy of tetrasodium EDTA as a catheter lock potentially shows that this agent could substantially reduce catheter-related infections and be used to treat patients with limited access.

Prevention of catheter related bloodstream infection by silver iontophoretic central venous catheters: a randomised controlled trial.

AIM: To evaluate the efficacy of silver iontophoretic central venous catheters in preventing catheter related colonisation and bloodstream infection among high risk patients in a tertiary hospital. METHODS: Patients requiring central venous access for a period greater than seven days were stratified into two groups according to systemic inflammatory response syndrome criteria before being randomly assigned to receive either silver iontophoretic or control catheters. The incidence of catheter colonisation and catheter related bloodstream infection (CRBSI) was recorded. RESULTS: Three hundred and four single lumen study catheters were inserted into 268 patients. Total duration of catheterisation was 5449 days (median, 12 days/catheter). Complete data could be evaluated in 270 catheters: 128 silver iontophoretic catheters and 140 untreated catheters. Forty seven silver iontophoretic catheters (36.7%) were colonised compared with 48 control catheters (33.8%). Seven cases (5.5%) of CRBSI occurred in patients who received silver iontophoretic catheters, compared with 11 cases (7.7%) in patients receiving control catheters. There was no significant difference in the incidence of catheter colonisation or CRBSI between silver iontophoretic and control catheters. When the duration of catheter placement was taken into consideration, Kaplan-Meier analysis showed no significant difference in the risk of CRBSI between the silver iontophoretic catheters and the untreated catheters (p = 0.77). CONCLUSION: There was no significant difference in the incidence of catheter colonisation or CRBSI among high risk patients between silver iontophoretic catheters and control catheters. Future prospective, randomised studies with a larger number of catheters are encouraged to confirm or refute these results.

Calcitonin precursors: early markers of gut barrier dysfunction in patients with acute pancreatitis.

BACKGROUND: Severe acute pancreatitis is associated with an early increase in intestinal permeability and endotoxemia. Endotoxin is a potent stimulator for the production and release of procalcitonin and its components (calcitonin precursors; [CTpr]). The aim of this study is to evaluate the role of plasma CTpr as an early marker for gut barrier dysfunction in patients with acute pancreatitis. METHODS: Intestinal permeability to macromolecules (polyethylene glycol 3350), serum endotoxin and antiendotoxin core antibodies, plasma CTpr, and serum C-reactive protein (CRP) were measured on admission in 60 patients with acute pancreatitis. Attacks were classified as mild (n = 48) or severe (n = 12) according to the Atlanta criteria. RESULTS: Compared with mild attacks of acute pancreatitis, severe attacks were significantly associated with an increase in intestinal permeability index (median: 0.02 vs. 0.006, P < 0.001), the frequency of endotoxemia (73% vs. 41%, P = 0.04), and the extent of depletion of serum IgM antiendotoxin antibodies (median: 43 MMU vs. 100 MMU, P = 0.004). Plasma CTpr levels were significantly elevated in patients with severe attacks compared with mild attacks on both the day of admission and on day 3 (median: 64 vs. 22 fmol/mL, P = 0.03; and 90 vs. 29 fmol/mL, P = 0.003 respectively). A positive and significant correlation was observed between the admission serum endotoxin and plasma CTpr levels on admission (r = 0.7, P < 0.0001) and on day 3 (r = 0.96, P < 0.0001), and between plasma CTpr on day 7 and the intestinal permeability index (r = 0.85, P = 0.0001). In contrast, only a weak positive correlation was observed between peak serum levels of CRP and plasma CTpr on admission (r = 0.3, P = 0.017) and on day 7 (r = 0.471, P = 0.049), as well as between CRP and each of the admission serum endotoxin (r = 0.3, P = 0.03) and the intestinal permeability index (r = 0.375, P = 0.007). CONCLUSIONS: In patients with acute pancreatitis, plasma concentrations of CTpr appear to reflect more closely the derangement in gut barrier function rather than the extent of systemic inflammation.

Calcitonin precursors in the prediction of severity of acute pancreatitis on the day of admission.

BACKGROUND: Calcitonin precursors are sensitive markers of inflammation and infection. The aim of this study was to evaluate the role of plasma calcitonin precursor levels on the day of admission in the prediction of severity of acute pancreatitis, and to compare this with the Acute Physiology And Chronic Health Evaluation (APACHE) II scoring system. METHODS: Plasma concentrations of calcitonin precursors were determined on admission in 69 patients with acute pancreatitis. APACHE II scores were calculated on admission. Attacks were classified as mild (n = 55) or severe (n = 14) according to the Atlanta criteria. Plasma calcitonin precursor levels were determined with a sensitive radioimmunoassay. RESULTS: On the day of hospital admission, plasma levels of calcitonin precursors were significantly greater in patients with a severe attack compared with levels in those with a mild attack of pancreatitis (median 64 versus 25 fmol/ml; P = 0.014), but the APACHE II scores were no different (median 9 versus 8; P = 0.2). The sensitivity, specificity, positive predictive and negative predictive values, and accuracy for the prediction of severe acute pancreatitis were 67, 89, 57, 93 and 85 per cent respectively for plasma calcitonin precursor levels higher than 48 fmol/ml, and 69, 45, 23, 86 and 50 per cent respectively for an APACHE II score greater than 7. Differences in the specificity and accuracy of the two prognostic indicators were significant (P < 0.001 and P = 0.001 respectively). A plasma calcitonin precursor concentration of more than 160 fmol/ml on admission was highly accurate (94 per cent) in predicting the development of septic complications and death. CONCLUSION: The assay of plasma calcitonin precursors on the day of admission to hospital has the potential to provide a more accurate prediction of the severity of acute pancreatitis than the APACHE II scoring system.

DNA fingerprinting analysis of coagulase negative staphylococci implicated in catheter related bloodstream infections.

AIMS: The epidemiological assessment of cases of coagulase negative staphylococcal catheter related bloodstream infection. METHODS: Two hundred and thirty patients with suspected catheter related bloodstream infection were evaluated over a two year period. Central venous catheters were cultured both endoluminally and extraluminally. Peripheral blood, catheter hubs, skin entry, and skin control sites were also cultured. Pulsed field gel electrophoresis (PFGE) was used to DNA fingerprint coagulase negative staphylococci isolated from patients with presumptive catheter related bloodstream infection. RESULTS: Sixty cases of catheter related bloodstream infection were identified, 21 of which were attributed to coagulase negative staphylococci. Two hundred and ninety four separate isolates of coagulase negative staphylococci from the 21 cases of catheter related bloodstream infection were subjected to PFGE (mean of 14 for each case). Catheter related bloodstream infection was only confirmed by PFGE analysis in 16 of the 21 cases because in the remaining five cases peripheral blood and central venous catheter coagulase negative staphylococci isolates were different. Skin entry, control skin, and central venous catheter hub isolates matched peripheral blood isolates in six, four, and seven cases, respectively. Coagulase negative staphylococci isolates could not be cultured from the patients' own skin in seven cases of catheter related bloodstream infection. Central venous catheter lumens were colonised in all cases of catheter related bloodstream infection compared with 44-81% of cases that had positive external surface catheter tip cultures, depending on the threshold used to define significant growth. CONCLUSIONS: Catheter related bloodstream infection as a result of coagulase negative staphylococci may be over stated in about a quarter of cases, unless a discriminatory technique is used to fingerprint isolates. No single, simplistic route of bacterial contamination of central venous catheters was identified, but endoluminal catheter colonisation is invariably present in cases of catheter related bloodstream infection.

Endotoxin, cytokines and lipid peroxides in children with intussusception.

BACKGROUND: Intussusception is a relatively common paediatric surgical emergency. The aim of this study was to investigate selected inflammatory mediators in children with acute intussusception and to identify potentially useful plasma markers of clinical outcome. METHODS: Clinical, radiographic, operative and pathological details were recorded prospectively of all children presenting to a single institution with a confirmed diagnosis of acute intussusception during 1 year. Paired acute and convalescent venous blood samples were collected in a standard manner for blinded analysis of the following: malondialdehyde, C-reactive protein (CRP), interleukin (IL) 6, neopterin, tumour necrosis factor alpha, endotoxin, and immunoglobulin (Ig) G and IgM antiendotoxin core antibody (EndoCAb). RESULTS: Thirty-two consecutive children (23 boys, nine girls) with a median age of 4 months were studied. Acute ileocolic intussusception was managed by air enema reduction (n = 19), operative reduction (n = 8) or surgical resection (n = 5). Peripheral blood cultures were sterile. Acute levels of plasma IL-6, neopterin and CRP were significantly raised in comparison to both normal laboratory ranges and convalescent samples (P < 0.001). Using stepwise discriminant analysis, CRP was identified as the best variable at distinguishing between the three treatment groups (P < 0.001). IgM EndoCAb concentrations were significantly greater in the convalescent sera of all the patients (P < 0.001). CONCLUSION: Acute ileocolic intussusception in childhood is associated with endotoxinaemia and significantly raised levels of circulating inflammatory cytokines. Plasma CRP at diagnosis showed a statistically significant positive correlation with disease severity.

In situ measurement of linezolid and vancomycin concentrations in intravascular catheter-associated biofilm.

We describe a new method for the measurement of antimicrobial concentrations in the biofilm associated with the endoluminal surface of intravascular catheters. We quantified endoluminal planktonic bacteria in haemodialysis catheters using the acridine orange method on catheter blood. After catheter removal, separate lumens were perfused in vitro with either vancomycin or linezolid to simulate in vivo antibiotic infusion. Biofilm was recovered using endoluminal brushes, weighed and assayed by fluoroimmunoassay for vancomycin and by bioassay for linezolid. Viable bacteria were counted by serial dilution and agar plating. Biofilm had measurable amounts of vancomycin in 11/11 catheter lumens post-infusion (0.3-18.2 mg biofilm per lumen, mean 6.8 mg; vancomycin concentration 0.2-89 mg/g biofilm, median 19 mg/g). By comparison, linezolid was detected in 4/11 catheter lumens post-infusion (0.5-18.1 mg biofilm per lumen, mean 5.9 mg; linezolid concentration 0.9-6.1 mg/g biofilm, median 1.5 mg/g). Percentage reductions in biofilm-associated bacterial counts post-antibiotic were 84-100%, median 95% (vancomycin) and 0-98%, median 91% (linezolid). We found a significant difference (P = 0.05; Wilcoxon rank sum test) in vancomycin concentrations in coagulase-negative staphylococcal biofilm (median 17.0 mg/g, mean 27.9 mg/g) compared with glycopeptide levels found in biofilm associated with other microorganisms (median 5.5 mg/g, mean 6.9 mg/g). Biofilm concentrations of vancomycin are generally higher than linezolid after antibiotic infusion, which can be explained partly by glycopeptide binding to glycocalyx. Neither antibiotic achieved consistent 100% kill of biofilm bacteria after single infusions, even when a very high concentration was present. The endoluminal brush technique can be used to measure antibiotic concentration in intravascular catheter-associated biofilm in situ. This approach can be exploited to measure biofilm antibiotic concentrations in vivo, without the need for catheter removal.

Rapid diagnosis of central-venous-catheter-related bloodstream infection without catheter removal.

BACKGROUND: Current methods for the diagnosis of bloodstream infection related to central venous catheters (CVC) are slow and in many cases require catheter removal. Since most CVC that are removed on suspicion of causing infection prove not to be infected, removal of catheters unnecessarily exposes patients to the risks associated with reinsertion. METHODS: The gram stain and acridine-orange leucocyte cytospin test (AOLC) is rapid (30 min), inexpensive, and requires only 100 microL catheter blood (treated with edetic acid) and the use of light and ultraviolet microscopy. We assessed the gram stain and AOLC test in suspected cases of catheter-related bloodstream infection, in comparison with two methods requiring catheter removal (tip roll and tip flush), and a third technique, done in situ (endoluminal brush) in conjunction with quantitative peripheral-blood cultures. FINDINGS: 128 cases of suspected catheter-related bloodstream infection were assessed in 124 adult surgical patients (median duration of CVC placement was 16 days). In 112 (88%) cases CVC blood was obtainable. Catheter-related bloodstream infection was diagnosed in 50 cases (culture of the same organism from the catheter, in significant numbers, and from peripheral-blood culture). The sensitivity of the gram stain and AOLC test was 96% and the specificity was 92%, with a positive predictive value of 91% and a negative predictive value of 97%. By comparison, the tip-roll, tip-flush, and endoluminal-brush methods had sensitivities of 90%, 95%, and 92%, and specificities of 55%, 76%, and 98%, respectively. INTERPRETATION: The gram stain and AOLC test is a simple, and rapid method for the diagnosis of catheter-related bloodstream infection. This diagnostic method compares favourably with other diagnostic methods, particularly those that require the removal of the catheter, and can permit early targeted antimicrobial therapy.

Evaluation of a novel endoluminal brush method for in situ diagnosis of catheter related sepsis.

AIMS: To determine the accuracy of a novel endoluminal brush method for the diagnosis of catheter related sepsis (CRS), which is performed in situ and hence does not require line sacrifice. METHODS: 230 central venous catheters in 216 patients were examined prospectively for evidence of CRS or colonisation using an endoluminal brush method in conjunction with peripheral blood cultures. The results were compared with those obtained using methods that require line sacrifice: extraluminal sampling (Maki roll) or endoluminal sampling (modified Cleri flush) of microorganisms. RESULTS: Only 16% of 128 patients suspected clinically of having line associated infection were confirmed as having CRS. In addition, 2 of 102 patients not suspected of having line associated infection had CRS. Line colonisation was apparent in approximately twice as many catheters using the Maki roll criteria (92%) compared with either the endoluminal brush (43%) or Cleri flush (43%). Furthermore, colonised catheters sampled using the Maki roll technique yielded mixed growth twice as often as when examined by endoluminal methods (17 and 8 cases, respectively). It was rare to detect either only endoluminal (4 of 22 episodes) or extraluminal (1 of 22 episodes) microorganisms in cases of CRS. In contrast, catheters defined as being colonised most frequently (59% of episodes) yielded only significant extraluminal growth. Only one case of CRS (5%) would have been "missed" if lines yielding a negative result from endoluminal brush sampling had been left in situ. Conversely, four episodes of CRS (18%) would not have been diagnosed by relying on extraluminal sampling alone. CONCLUSIONS: Diagnosis of CRS by the endoluminal brush method can be achieved without line sacrifice and is more sensitive (95%) and specific (84%) than extraluminal sampling of the catheter tip by the Maki roll technique (82% and 66%, respectively).

Rapid diagnosis of catheter-related sepsis using the acridine orange leukocyte cytospin test and an endoluminal brush.

BACKGROUND: In neonates, the acridine orange leukocyte cytospin (AOLC) test has been found to be a highly sensitive test for the detection of infected i.v. catheters in situ, which provides a result in less than 1 hour. Preliminary data suggested that the AOLC test was of limited value in adults. We report here a modification of the test for adult patients with indwelling central venous catheters. METHODS: A prospective study was performed on two groups of 50 adult patients with suspected sepsis and a central venous catheter. The AOLC test was carried out after the clinical decision to remove the catheter had been made. In group 1 patients, a blood sample was withdrawn from the catheter for the AOLC test. In the patients in group 2, an endoluminal brush was used to "sweep" the catheter before the collection of the blood sample. Results of the AOLC test were compared with culture of the removed catheter tip. RESULTS: From the catheters in group 1 (no brush), 17 catheter tips were found to be infected, but the AOLC was positive in only two patients (12%). In group 2 (brush), 18 tips were infected, and the AOLC test was positive in 15 patients (83%). The use of the endoluminal brush significantly improved the yield of the AOLC test (p < .01) to levels reported in neonates. The AOLC test produced no false positives in either group CONCLUSION: When used independently, the AOLC test was not sensitive enough to detect catheter-related sepsis. However, in combination with an endoluminal brush, the AOLC test was much more sensitive and has the potential to provide a simple, rapid, and accurate diagnostic test for catheter-related sepsis, which does not require removal of the catheter.

PCR for the detection and typing of campylobacters.

The flaA gene of Campylobacter sp. was amplified using PCR. Primers were chosen which amplified 1.3 kb of the flaA gene in Camp. jejuni and Camp. coli. 'Campylobacter upsaliensis' amplimer was approximately 1.7 kb in size and was easily distinguishable. Other species of campylobacter failed to yield amplimer. The amplimer was digested with Alu 1 which demonstrated considerable restriction fragment length polymorphism and should allow the development of a rapid novel typing scheme which does not rely on previous culture of campylobacter strains.

Rapid diagnosis of central venous catheter sepsis.

Signs of infection with a central venous access device in situ raise the possibility of catheter sepsis. We evaluated three tests for diagnosis of infection in infants with suspected catheter sepsis. The acridine orange leucocyte cytospin (AOLC) test was 87% sensitive and 94% specific in the diagnosis of catheter-related sepsis defined by quantitative blood culture. The C-reactive protein and nitroblue tetrazolium tests were not as useful. Using the AOLC results, available in an hour, we now remove fewer catheters on suspicion of sepsis alone.