The cost-effectiveness of ESBL detection: towards molecular detection methods?

Correct detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control and antibiotic choice. We performed a study to determine the cost-effectiveness of phenotypical testing, which can be inaccurate, and genotypical tests, which are considered to be more reliable but also more expensive. All patients that had been in isolation in the Amphia hospital because of the detection of ESBL according to the ESBL Etest were included in the survey. All strains were retested using the double disk confirmation test (DDCT) and a genotypical method. This was a commercially available microarray (Check-Points). Discordant results were confirmed by PCR and sequencing. In total 174 patients were included. In 24 of 174 (14%) patients, ESBL carriage could not be confirmed with the microarray. This was verified with PCR and sequencing. The mean duration of isolation was 15 days, adding up to a total number of isolation days of 2571. False-positive results according to the microarray resulted in a total of 279 days of unnecessary isolation for the Etest and 151 days for the DDCT. Using Etest to detect the presence of ESBL results in a false-positive outcome in 14% of the cases. This results in unnecessary isolation of patients, which can be omitted by using a genotypic method.

Severe prosthetic joint infection in an immunocompetent male patient due to a therapy refractory Pseudallescheria apiosperma.

Prosthetic joint infections (PJI) are rarely due to fungal agents and if so they are mainly caused by Candida strains. This case represents a PJI caused by a multi-drug resistant Pseudallescheria apiosperma, with poor in vivo response to itraconazole and voriconazole. This case differs also by the way of infection, since the joint infection did not follow a penetrating trauma. In the majority of cases, Scedosporium extremity infections remain local in immunocompetent individuals. We report a persistent joint infection with multiple therapeutic failures, and subsequent amputation of the left leg. Detailed clinical data, patient history, treatment regime and outcome of a very long-lasting (>4 years) P. apiosperma prosthetic knee infection in an immunocompetent, 61-year-old male patient are presented with this case. The patient was finally cured by the combination of multiple and extensive surgical interventions and prolonged antifungal combination therapy with voriconazole and terbinafine.

Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material.

We developed a dermatophyte-specific single-tube real-time PCR assay based on internal transcribed sequences. This assay allows the rapid detection and identification of 11 clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within a few hours. Analysis of 145 clinical samples (107 nail, 36 skin scale, and two hair) by both real-time PCR and a PCR-reverse line blot (PCR-RLB) assay described earlier revealed that 133 of the 145 samples had concordant real-time PCR and PCR-RLB detection results (83 positive, 49 negative, and one inhibited). Six samples were positive by real-time PCR and negative by PCR-RLB, and two were negative by real-time PCR and positive by PCR-RLB. Four samples demonstrated inhibition in one of the two PCR assays. Only one of 83 positive samples had discordant identification results between both assays (Trichophyton verrucosum and Trichophyton erinacei by real-time PCR and Trichophyton erinacei by PCR-RLB). Dermatophytes present in seven positive samples that were incompletely identified as Trichophyton sp. by PCR-RLB were identified to the species level by real-time PCR as Trichophyton interdigitale and Trichophyton rubrum in six cases and one case, respectively. One hundred and twenty of 145 samples were also analysed by conventional dermatophyte culture and by direct microscopy. Our single-tube real-time PCR assay proved to be suitable for direct detection and identification of dermatophytes in nail, skin and hair samples with minimal total assay time (4 h after overnight lysis) and hands-on time, without the need for post-PCR analysis, and with good sensitivity and specificity.

Validation of PCR-reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples.

A dermatophyte-specific PCR-reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.

Decolonization of methicillin-resistant Staphylococcus aureus using oral vancomycin and topical mupirocin.

The objective of this study was to assess the efficacy and safety of a short course of oral vancomycin and intranasal mupirocin ointment in the eradication of methicillin-resistant Staphylococcus aureus (MRSA) colonization. During an outbreak of MRSA, the colonized subjects received oral vancomycin and topical mupirocin. They were screened for MRSA 1, 3, 6 and 12 months after decolonization. A questionnaire was developed to evaluate the side-effects of oral vancomycin. Thirty-five subjects were treated. Clearance was achieved in all cases, in 24 (69%) subjects after one course of therapy. Twenty-eight (80%) subjects experienced some side-effects, including six (17%) who did not tolerate oral vancomycin. Although oral vancomycin, in combination with topical mupirocin, is effective in the elimination of MRSA colonization, there is a need for further studies to confirm our results and to evaluate the safety of oral vancomycin.

Search for enterotoxin gene in Bacteroides fragilis strains isolated from clinical specimens in Poland, Great Britain, The Netherlands and France.

BACKGROUND: Bacteroides fragilis is a member of normal human flora and well known pathogenic agent. This bacterium produces many virulence factors. In 1984 new virulence factor--enterotoxin was described. The aim of the study was to search for enterotoxin gene in B. fragilis strains isolated from clinical specimens. MATERIAL AND METHODS: Strains isolated in Poland, Great Britain, France and the Netherlands were cultured on BBE medium. For DNA isolation Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland) was used. In order to detect enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied utilizing the following primers: 404 (GAG CGG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in thermocycler Techne. The amplification products were detected by the electrophoresis in 1% agarose gel. RESULTS: Among 65 investigated B. fragilis strains, the enterotoxin gene was detected in DNA isolated from 12 strains. CONCLUSION: The enterotoxin producing B. fragilis strains were detected among strains isolated from different clinical specimens in Poland, Great Britain, the Netherlands and France.

[Shifts in the species spectrum of mycoses in The Netherlands from 1970-1990]. / Verschuivingen in het soortenspectrum van mycosen in Nederland in het tijdvak 1970-1990.

A review is presented of yeast and mould infections occurring in humans in the Netherlands. The occurrence of the dermatophytes Trichophyton rubrum and T. mentagrophytes tends to increase, while Microsporum canis and particularly Epidermophyton floccosum have become less common. The yeast Candida glabrata is particularly often involved in infections of the urinary tract. Candida krusei, C. parapsilosis and C. tropicalis have become less significant. Remarkable differences are found between the spectra of Aspergillus species causing infections in lungs and in ears; an entirely different pathogenesis is to be presumed. The number of systemic mycoses in the Netherlands is underestimated. The possibility of hundreds of cases each year cannot be excluded.

Prevention of group B streptococci transmission during delivery by vaginal application of chlorhexidine gel.

In a prospective study in 227 parturients, carriership of group B streptococci was established to be 25%. In carriers, transmission of streptococci to the newborn occurred in 50%. 10 ml of a chlorhexidine gel containing hydroxypropylmethylcellulose was introduced into the vagina during labor in 17 parturients, who were known to be carriers of group B streptococci from the first trimester of pregnancy. In none of the newborns from these mothers colonization by group B streptococci did occur. Vaginal application of chlorhexidine may prevent transmission of group B streptococci, and serve as an alternative to intrapartum prophylaxis using antibiotics. A large multicenter randomized controlled study should be performed to confirm this hypothesis.

Imipenem as monotherapy in the treatment of intensive care patients with severe infections.

In an open study, 24 intensive care patients were treated with imipenem/cilastatin as monotherapy for serious bacterial infections. Twenty-one patients were treated for bronchopulmonary infection, two patients for septicaemia, and one patient for an empyema. Initially all strains were susceptible to imipenem. Gram-negative bacilli accounted for 80% of these isolates. The most frequently isolated species were Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa. All 24 patients were considered clinically cured. Sixteen of these patients (67%) were both clinically and microbiologically cured. In eight of the 24 patients (33%), the strains isolated initially persisted. In eight of the 24 patients (33%), colonization of the respiratory tract developed. Two of the five Ps. aeruginosa isolates developed resistance during therapy but in none of these patients was therapy considered to have failed. In 12 patients (50%), transient elevations in hepatic function tests were observed and these were probably drug-related. The present study supports the view that imipenem/cilastatin may be useful as monotherapy in the treatment of severe infections in intensive care patients.