Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis.

AIM: Introduction of a protocol for broad-range diagnosis of bacterial infections, which remain negative in culture. METHODS AND RESULTS: The new TaqMan real-time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 mul EDTA-blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24.3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. CONCLUSIONS: The introduced broad-range real-time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing-point values and with the Blast result of both the sample and the controls. SIGNIFICANCE AND IMPACT OF THE STUDY: This work introduces a new and well-evaluated broad-range real-time PCR protocol for diagnosis of bacterial infections.

Simplifying the WHO 'how to hand rub' technique: three steps are as effective as six-results from an experimental randomized crossover trial.

OBJECTIVES: The World Health Organization (WHO) issued guidelines on hand hygiene recommending a six-step 'how to hand rub' technique for applying alcohol-based hand rub. However, adherence to all six steps is poor. We assessed a simplified three-step technique and compared it to the conventional WHO six-step technique in terms of bacterial count reduction on healthcare workers' hands. METHODS: Thirty-two participants were randomly assigned to clean their hands following the six-step 'how to hand rub' technique (WHO reference group) or a simplified three-step technique (intervention group). Assignments were reversed after 1 day. The degree of bacterial killing was assessed following the European norm for testing hand hygiene products. Hands were contaminated with Escherichia coli, and the mean logarithmic reduction in bacterial counts was compared between both techniques. RESULTS: Bacterial density before hand hygiene performance did not differ between the WHO reference group (median 6.37 log10 CFU, interquartile range (IQR) 6.19-6.54) and the intervention group (median 6.34 log10 CFU, IQR 6.17-6.60, p 0.513). After hand hygiene, the logarithmic reduction factor was higher in the intervention group (median 4.45, IQR 4.04-5.15) compared to the WHO reference group (median 3.91, IQR 3.69-4.62, p 0.021). CONCLUSIONS: The WHO six-step 'how to hand rub' technique can be simplified to a 3-step procedure based on the reduction of bacterial counts on healthcare workers' hands achieved under experimental conditions. The proposed technique is easier to perform and could improve adherence to the execution of hand hygiene action.

Surgical hand disinfection with alcohols at various concentrations: parallel experiments using the new proposed European standards method.

OBJECTIVES: To establish the concentration of isopropanol that exerts the same immediate and sustained effects as n-propanol 60% v/v in surgical scrubbing, and to assess the performance of the test method proposed as the European standard in parallel experiments. DESIGN: Isopropanol at concentrations of 70%, 80%, and 90% v/v was tested in comparison with n-propanol 60%, the proposed reference preparation, in the draft method proposed by the European standard. A Latin square design was used with four balanced blocks of five volunteers each in four experimental runs that were spaced by intervals of 1 week each. Volunteers were allotted randomly to one of the four blocks. Independently, the volunteers' right and left hands also were randomized into two groups for the assessment of either immediate or sustained effects. SETTING: Two laboratories supervised by two investigators, one from Vienna, Austria, and one from London, The United Kingdom. METHOD: The release of skin flora from the fingertips of clean hands was assessed before and after treatment by immediate sampling from one hand and by sampling of the other, gloved hand after 3 hours. The mean log10 reductions (RF) of bacterial release achieved by rubbing the alcoholic preparations for 3 minutes onto the hands were established. RESULTS: For both experiments, the immediate effects of isopropanol 70% (RF, 2.0 and 2.1, respectively) were significantly smaller than those of the reference n-propanol 60% (RF, 2.4 and 2.6, respectively). This also was found with the sustained effects (RF, 0.7 and 1.1 vs 1.0 and 1.6, respectively). At 90%, isopropanol equalled the immediate effect of n-propanol 60%, whereas at 80% it proved slightly (although not significantly) less active. There were no significant differences in the results of both investigators. The sustained effects of isopropanol 80% and 90% were both larger than the reference in Vienna but were found smaller by the London investigator; none of the differences were significant. Mean RFs were significantly different between Vienna and London with n-propanol 60% and isopropanol 70%, but not with isopropanol at 80% or 90%. CONCLUSIONS: At 90%, isopropanol is as effective as n-propanol 60%, which was proposed by the European Committee for Standardization as a reference in testing products for surgical hand disinfection. It could, therefore, serve as an alternative if the proposed agent is undesirable for any reason. In parallel experiments by two investigators, the proposed test method proved well workable; the results were very similar and the conclusions identical.

Comparison of ELISA antigen preparations alone or in combination for serodiagnosing Helicobacter pylori infections.

The immunoglobulin G antibody response to Helicobacter pylori was assessed in 78 patients with non-ulcer dyspepsia using five different antigen preparations. All patients were endoscoped and biopsied. The H pylori state was determined histologically on at least two endoscopic biopsy specimens using a modified Giemsa stain. The ultracentrifuged cell sonicate, acid glycine extract, and 120 kilodalton protein antigens were specific in diagnosing infection (95-98%), but had only moderate sensitivity (70-84%). By mixing either of the two complex antigens with the 120 kilodalton protein, the sensitivity of the test was increased to 97% without affecting the high specificity. The combination of ultracentrifuged sonicate or acid glycine extract with the 120 kilodalton protein therefore seems to be superior to the individual antigen preparations and is particularly suitable for the serodiagnosis of H pylori infection.

European norms in hand hygiene.

In the area of hand hygiene, European Norms exist, or are under development, with regard to protective gloves and for assessing the antimicrobial efficacy of hand disinfectants. Important norms for gloves are EN 420 (General requirements), EN 374 (Protective gloves against chemicals and microorganisms) and EN 455 (Medical gloves for single use). A suspension test for the demonstration of bactericidal activity (prEN 12054) is obligatory for hand disinfectants in all fields of application; a test to prove activity against yeasts applies only to hygienic hand rub. (Optional) Claims for virucidal activities can be substantiated by prEN 1476 and, in future, for mycobactericidal capacity by a test which is still under development. In vivo tests exist for post-contamination treatments, hygienic hand wash and hygienic hand rub (EN 1499 and EN 1500 respectively), and for the preoperative surgical hand rub/wash (prEN 12791). The two former tests employ artificially contaminated hands, the latter test is done with clean hands. All in vivo tests use reference hand treatments (with unmedicated soap or 2-propanol 60% (vol.) or 1-propanol 60% (vol.), respectively) against the results of which are compared with those achieved with the product under test and with the same volunteers. An antiseptic soap needs to be significantly more efficacious than unmedicated soap, a product for hygienic hand rub must not be inferior to the reference treatment with 2-propanol, and a surgical hand disinfectant must not cause a smaller bacterial reduction than the reference preparation with 1-propanol, immediately, and after 3 h. An (optional) claim for sustained activity of a surgical disinfectant needs to be demonstrated by achieving a significantly stronger bacterial reduction after 3 h than the reference preparation.

Arguments for alcoholic hand disinfection.

The non-aqueous use of ethanol or propanols offers various advantages over washing hands with either unmedicated or medicated soap in both hygienic and surgical hand disinfection. Alcohols exert the strongest and fastest activity against a wide spectrum of bacteria and fungi (but not bacterial spores) as well as enveloped (but less so against non-enveloped) viruses, being little influenced by interfering substances. They are of low toxicity and offer acceptable skin tolerability when made up with suitable emollients. The mode of their application is simple and three to four times more economical of time than wash procedures, features which help to increase the compliance with the rules of hand hygiene.

Test models for hygienic handrub and hygienic handwash: the effects of two different contamination and sampling techniques.

Two methods for artificial contamination of hands and two sampling techniques to recover the test organisms were compared for their effects on the results of two post-contamination hand treatments: a handrub with two portions of 3 ml of 2-propanol 60% v/v for 1 min, and a handwash with liquid soap 20% w/v for 1 min followed by a 15 s rinse. The two contamination methods involved a short immersion of the hands (up to the middle of the mid-hand) in a suspension of the test organism followed by either air-drying (3 min) or drying by rubbing the hands' vigorously against each other (3 min) in a standardized way. The two sampling techniques consisted of rubbing the fingertips in either 10 ml trypticase soy broth (TSB) against the bottom of a Petri dish; or 100 ml TSB against glass beads contained in a bowl. Sixteen volunteers were randomly allotted to four blocks of four. They carried out the four possible combinations of two treatments and two contamination methods in a series of four tests arranged in a Latin-square design. In addition, the two sampling techniques were compared with each other concurrently by sampling of the right and left hand each with a different one of the two techniques. The alcoholic handrub reduced the release of test organisms significantly (2P less than 0.005) more effectively, by 1.1-1.3 x log10, than did the handwash with liquid soap, regardless of the contamination or sampling method. Whereas the two recovery techniques yielded virtually identical results in corresponding situations, the method of artificial contamination affected the mean reduction factors, strongly.(ABSTRACT TRUNCATED AT 250 WORDS)

Surgical hand disinfection: effect of sequential use of two chlorhexidine preparations.

The antimicrobial efficacy of three 'two-phase' surgical hand disinfection procedures was compared, in a volunteer study, to 60% n-propanol, applied for 5 min, which is the reference hand-disinfection procedure used in Austria and West Germany (FRG). The procedures involved sequential use of unmedicated soap or a disinfectant-detergent containing 4% chlorhexidine gluconate (CHX; 'Hibiscrub') followed by a handrub preparation containing 70% w/w isopropanol plus 0.5% CHX ('Hibisol'). The immediate and sustained effects (3 h) of washing with unmedicated soap (3 min) followed by rubbing on 'Hibisol' (4 min) were significantly smaller (log10 reductions of 1.72 and 1.12) than with each of the other procedures. Use of 'Hibiscrub' (3 min) and 'Hibisol' (4 min) produced log10 reductions of 2.50 and 1.71, equalling those of the reference procedure with n-propanol (2.49 and 1.78). When 'Hibisol' was used for 5 min rather than 4 min, a considerable, though not significant, increase in effect was achieved (log10 reductions of 2.90 and 2.07). Replacement of unmedicated soap by 'Hibiscrub' could significantly improve the effectiveness of the hand disinfection procedure commonly used by surgeons in German-speaking countries; namely to wash hands first with soap and then disinfect them with an alcoholic preparation. It may also be of additional advantage as this adds another 'layer' of CHX when 'Hibisol' rather than alcohol alone is used.

Effect of chlorhexidine-containing detergent, non-medicated soap or isopropanol and the influence of neutralizer on bacterial pathogenicity.

In handwashing experiments with Salmonella typhimurium the effect of chlorhexidine (CHX) on the pathogenicity of surviving bacteria was assessed with and without a neutralizer in a mouse model of infection. Without neutralizer the LD50 of CHX handwash fluids was raised. Neutralizer in suspensions of untreated bacteria caused a reduction of LD50 up to 1.2 logs. Thus, in contrast to soap or alcohol, CHX without neutralizer exerted a slight 'depathogenizing' action and neutralizer a slight 'pathogenizing' effect in the experimental model used. However, in comparison to the efficiency of handwashing procedures which reduce the number of bacteria available for transfer by at least 3.0 to 4.2 logs, the size of these effects seems to be negligibly small and unpredictable. Therefore, the single most important parameter in assessing the potency of disinfectants remains the reduction of viable counts with time.

Do 'chlorine covers' exert a sustained bactericidal effect on the bacterial hand flora?

Treatment of skin with chlorine generates 'chlorine covers' which, in a previous study, exerted significant sustained bactericidal effects against transient skin flora on the upper arm and forearm. In this investigation, this effect was studied on both the transient and resident flora of the hands using test models for the evaluation of hand disinfectants as agreed upon in Austria and Germany. Chlorine covers were generated by bathing hands in a solution of 2% sodium tosylchloramide for 1 min. Subsequently, this cover was destroyed on one, randomly selected, hand by bathing it in a solution of 0.5% sodium thiosulphate for 15 s. The amount of chlorine on the fingertips of chlorinated hands was 2.2 +/- 0.4 micrograms/cm2; that on subsequently dechlorinated hands was 0.2 +/- 0.1 microgram/cm2. In experiments with artificially contaminated hands (Escherichia coli), the kinetics of bacterial die-off were the same on both hands. Also, in experiments with resident flora, the kinetics of bacterial die-off did not suggest bacterial reductions that increase with the duration of contact with the chlorine cover. It was concluded that with the test models used, a significant sustained antimicrobial effect of the chlorine cover could not be demonstrated on hands.

The influence of cosmetic additives on the acceptability of alcohol-based hand disinfectants.

A prospective, randomized double-blind study with intra-individual comparison of the results was undertaken with 20 volunteers to assess the influence of cosmetic additives on the acceptability of a mixture of n-propanol (50% v/v) and isopropanol (30% v/v) for hand disinfection. Three to 5 ml of antiseptic was rubbed into the hands until dry 15 times a day, 5 days a week and for 2 weeks per preparation. For self-assessment the parameters 'appearance', 'intactness', 'turgor' and 'sensation' were evaluated weekly by visual analogue; for assessment by a dermatologist the same parameters except 'sensation' were used. Each score was compared before and after treatment. The antimicrobial efficacy of the alcoholic mixture was equivalent to or better than the standard (isopropanol 60% v/v, 1 min). The frequent application of these antiseptic preparations caused a slight but significant deterioration of the skin condition as judged by both self-assessment and dermatologist; however, this was significantly less when the antiseptic contained cosmetic additives. It is concluded that the addition of suitable emollients can significantly increase the acceptability of alcoholic disinfectants.

A comparison of the effects of preoperative whole-body bathing with detergent alone and with detergent containing chlorhexidine gluconate on the frequency of wound infections after clean surgery. The European Working Party on Control of Hospital Infections.

In a prospective, randomized, double-blind, placebo-controlled study involving 27 surgical units in six European countries, the effect of preoperative whole-body bathing on two occasions with a detergent containing chlorhexidine (CHX+) on the incidence of wound infection in elective, clean surgery was compared with two bathings with a detergent without chlorhexidine (CHX-). In the CHX+ group 2.62% of 1413 patients and in the CHX- group 2.36% of 1400 patients subsequently became infected. The infection rate in the CHX+ group was 1.11 times that in the CHX- group with 95% confidence limits ranging between 0.69 and 1.82. Consequently, bathing patients twice preoperatively with chlorhexidine-detergent did not reduce the incidence of infection of clean wounds.

Serological tests for monitoring Helicobacter pylori eradication treatment.

Serological tests for the detection of Helicobacter pylori-specific antibodies are useful in epidemiological studies, as a pre-endoscopic screening procedure, and as therapeutic follow-up. For the latter application, they represent an alternative to invasive and expensive diagnostic methods such as endoscopy and breath test, respectively. However, serological tests are suitable only for long-term treatment monitoring in adults. As the significant reduction of specific antibodies, and not seronegativity by itself, is regarded as a criterion of therapeutic success, it is always necessary to establish the antibody kinetics. Enzyme-linked immunosorbent assay (ELISA) based tests are best suited for this. The most relevant antibody class to be detected is H. pylori-specific IgG. The main indication for employing serology is the therapeutic follow-up of patients who, on the whole, are free of complaints.

Amoxicillin for the treatment of Helicobacter pylori infection.

Amoxicillin is one of the most active antimicrobials against Helicobacter pylori in vitro, with a minimum inhibitory concentration (MIC) of < or = 0.01-0.1 mg/l. Thus far, neither primary nor secondary resistant strains have been found. Amoxicillin, which has a bactericidal effect on H. pylori, but is less inhibitory in the stationary growth phase and against cell-adherent or slowly growing H. pylori, probably has both topical and systemic activity. It is fairly acid stable and is less affected by gastric acidity than macrolides. Nevertheless, its activity in vivo is considerably enhanced when it is given concomitantly with proton pump inhibitors. Several amoxicillin-containing treatment regimes have yielded H. pylori eradication rates of > or = 90%. Of particular interest are 1-week treatment regimens containing amoxicillin + clarithromycin + omeprazole, or amoxicillin + metronidazole + omeprazole, as well as a 1-h topical therapy developed in Japan.

A new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceae.

A new selective differential agar medium for rapid presumptive identification of Enterobacteriaceae from water and food samples is described (EMX ID agar). By a combination of fluorogenic and chromogenic substrates, the medium detects the presence of beta-D-glucuronidase, beta-D-galactosidase, beta-D-xylosidase, tryptophane deaminase and H2S; additionally, cytochrome-oxidase and indole production can be demonstrated. This medium provides an inexpensive means for simple and rapid presumptive identification of E. coli and coliforms and for the differentiation within the Klebsiella-Enterobacter and the Proteus-Providencia-Morganella group. Furthermore, it allows to distinguish between the H2S-positive Enterobacteriaceae Citrobacter freundii, Salmonella spp., S. arizonae, Edwardsiella, Proteus mirabilis, P. vulgaris and some oxidase-positive bacteria.

[Infection with Chlamydia trachomatis in patients of an ambulatory sterility clinic]. / Infektionen durch Chlamydia trachomatis bei Patientinnen einer Sterilitätsambulanz.

In order to investigate a presumed association of certain anamnestic data with Chlamydia trachomatis infection of infertile women, appropriate specimens were examined from 100 patients of an infertility clinic. PCR and nucleic acid hybridization were positive in 5% of the patients, corresponding in both tests; IgG was found in 31% and IgA in 5% of patients. The prevalence was highest in the age-group of 26-35 years (21 of 55) and higher in patients who had been trying to conceive for > 2 years (12 of 37) as than those trying < or = 2 years (3 of 13). The proportion of IgG-positive women was significantly (p = 0.03) smaller in Turkish patients (3 of 22) than in those of other origin (11 of 33 from former Yugoslavia and 15 of 39 Austrian women). The prevalence was higher in patients with reported pelvic inflammatory disease (11 of 30) as compared to patients without (15 of 50). With a pathological state of the Fallopian tubes the prevalence was 12 of 25, with normal tubes 6 of 18 (p > 0.05). Thus, there seem to exist anamnestic hints as to chlamydial infections. Because of their high prevalence in patients with sterility we recommend screening for chlamydial infection prior to undertaking any invasive diagnostic procedure of the Fallopian tubes.

The efficacy of antimicrobial treatment in Campylobacter pylori-associated gastritis and duodenal ulcer.

The efficacy of various antimicrobial and anti-ulcer agents on the elimination of Campylobacter pylori in duodenal ulcer patients was investigated. Ranitidine, cimetidine, pirenzepine, aluminium phosphate gel as well as combinations of H2-receptor antagonists or pirenzepine + penicillin V, ciprofloxacin, ofloxacin, phenyl-mercuryborate or rifampicin had no influence on C. pylori in vivo. Short term elimination of C. pylori was achieved in 3/15 patients treated with ranitidine + bacampicillin and in 1/5 treated with cimetidine + metronidazole. This elimination was accompanied by a significant reduction of polymorphonuclear infiltration of the antral mucosa. Development of bacterial resistance was observed in patients with additional quinolones, metronidazole and rifampicin but not in patients treated with betalactam antibiotics.