A new agar screen plate for extended-spectrum β-lactamase (ESBL) detection was evaluated with 50 clinical isolates of ESBL-producing Enterobacteriaceae species: Enterobacter cloacae (n = 10), Escherichia coli (n = 10), Klebsiella oxytoca (n = 3), Klebsiella pneumoniae (n = 25), and Proteus mirabilis (n = 2). Fecal samples were artificially inoculated with 2 concentrations (25 and 250 colony forming units [CFU]/plate) of the test strains and then applied to the new agar screen plates. By this approach, the new agar formula detected growth that was suggestive of ESBL activity in 44 of 50 (88%) and 50 of 50 (100%) of ESBL strains with 25 and 250 CFU/plate, respectively. A limitation of the agar screen plates was a lack of some specificity. Among 15 strains with resistant phenotypes other than ESBL (K1 producers of K. oxytoca, 6 strains; 9 strains with AmpC phenotype), growth was recorded in 7 (25 CFU/plate) and 11 (250 CFU/plate) of 15 strains. In conclusion, the new agar screen plate is a sensitive and convenient method to directly screen for ESBL organisms in rectal swabs or stool samples, with the potential for incorporation into routine clinical laboratory service.

BACKGROUND:Elevated antibodies against Chlamydia pneumoniae have been associated with coronary artery disease. In patients undergoing percutaneous coronary angioplasty, we therefore investigated the effect of roxithromycin on symptomatic restenosis and determined antichlamydial antibodies as well as inflammatory and immunological parameters.METHODS:A total of 327 patients undergoing coronary angioplasty were randomized to roxithromycin or placebo and followed-up for 1 year. Antibodies were determined by microimmunofluorescence and enzyme-linked immunosorbent assay; C-reactive protein, interleukin-10, tumor necrosis factor-alpha (TNF-alpha), and eotaxin were determined by enzyme-linked immunosorbent assay.RESULTS:Although the frequency of restenosis was not affected by roxithromycin (25 restenoses vs 32 in the control group), antichlamydial antibodies increased during follow-up (anti-CP IgG +12 +/- 2%, P < .001). Concentrations of TNF-alpha and eotaxin increased as well (TNF-alpha +9 +/- 1% and eotaxin +10 +/- 2%) and correlated with antichlamydial antibody concentrations (TNF-alpha, r = 0.23, P = .02; eotaxin, r = 0.32, P = .002).CONCLUSIONS:Treatment with roxithromycin was not associated with a reduction of symptomatic restenoses. During follow-up, a marked increase in antichlamydial antibodies, TNF-alpha, and eotaxin was observed, suggesting that angioplasty-induced plaque rupture induces a specific immunological response without activation of inflammatory mechanisms as represented by C-reactive protein. Whether this mechanism occurs in all plaque ruptures remains to be determined.

ABSTRACT The case of a severe spinal osteomyelitis in a 3- year- old rhinoceros homed viper, Bitis nasicornis, is presented. The snake was captive-born and kept by the author since the age of three months with no history of trauma. Despite massive destruction of the spine with compression of the spinal cord, the snake showed no altered behavior. The only clinical signs were movement restrictions of the afflicted body region and a swelling which became obvious shortly before the animal was euthanatized. Enterococcus spp. were found to be the causative agent of the infection.

It is important to shorten the window period after acute HIV infection in which infected individuals are still antibody-negative, especially in blood donors. Newly developed fourth-generation assays detect antibodies to HIV-1, including subtype O, and to HIV-2 and, simultaneously, p24 antigen of HIV-1. To evaluate this assay for daily routine work we compared it with different third-generation assays using sera from uninfected patients and patients with known HIV infection. The most interesting sera are those drawn during seroconversion from freshly infected patients. Whenever we encounter such a patient with acute HIV infection we store the serum in aliquots at -20 degrees C. Thus, we were able to establish our own seroconversion panel and use it in our laboratory for evaluation of new assays. The new test was shown to be able to detect all chronically HIV-infected individuals and four of six patients during seroconversion although in two of these patients conventional assays for HIV antibodies were still negative. The rate of unspecific reactivities was slightly higher as compared with third-generation assays.

Objective: Patients on maintenance hemodialysis are known to have an elevated risk of acquiring hepatitis C virus (HCV) infection. Therefore, a reliable diagnosis of HCV infection is essential in order to prevent the spread of the disease in dialysis units. However, whether PCR examination is dispensable in hemodialysis patients has been debated. Methods: From 1995 to 2002, serum samples from all hemodialysis patients at our hospital (n = 1,774) were screened by serological assays and by polymerase chain reaction (PCR). Results: In 25 of these patients acute HCV infection was observed and in 11 patients HCV seroconversion was delayed for 3–16 months. During this time the infection was exclusively detectable by PCR. Conclusion: Despite the growing demand for cost-effectiveness in the health system, HCV PCR examination must remain an essential part of the routine screening in hemodialysis patients.

The reverse transcriptase V207I mutation within the hepatitis B virus (HBV) polymerase is associated with resistance to lamivudine in vitro. The prevalence of this mutation in treatment-naive patients was 1% (1/96). A follow-up of the patient carrying this mutation prior to treatment revealed no loss of sensitivity of HBV to lamivudine in vivo.

Mycobacterium haemophilum has rarely been implicated in human disease. The organisms have been isolated mainly in patients with human immunodeficiency virus (HIV) disease or transplant recipients. We describe the first case of a disseminated M. haemophilum infection as initial manifestation of AIDS in Europe.

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 2041 mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P =.006) and rt180M mutants (81% in group A vs 40% in group D, P =.015) prevailed in group A. The median time of shift from rt2041 to rt204V mutants was significantly shorter in group A (4 months in group A, > 12 months in group D, P <.001). Additional resistance associated mutations were detected exclusively in group D (P =.004). In a multivariate analysis, HBV genotype (P =.039) and pretreatment serum HBV DNA (P =.001) were independently associated with emerging rt2041 or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log(10) 6.99 copies/ml; range 3-9) compared with group D (mean log(10) 6.1 copies/ml; range 3.3-8; P =.04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV.

ABSTRACT Reptile-associated Salmonella infections are an increasing problem for humans. We have prospectively screened two breeding groups of 16 pet snakes for colonization with Salmonella species. Various serovars of S. enterica subsp. diarizonae were found in 81% of the snakes. To avoid transmission, strict hygienic precautions should be applied when reptiles are handled.

OBJECTIVE:We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species.METHODS:Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6).RESULTS:The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase.CONCLUSION:The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.

OBJECTIVE:In recent years, cefotaximases of the CTX-M type have become a predominant cause of resistance to extended-spectrum cephalosporins in Gram-negative bacteria. Although most enzymes provide higher levels of resistance to cefotaxime than to ceftazidime, mutants with enhanced catalytic efficiency against ceftazidime have recently been described. This report identifies another ceftazidime-resistant mutant of the CTX-M class of enzymes.METHODS:Two ceftazidime-resistant strains, Escherichia coli IFI-1 and Klebsiella pneumoniae IFI-2, were isolated from a 46-year-old man during treatment of postoperative peritonitis with ceftazidime. Susceptibility testing, mating-out assays, isoelectric focusing as well as PCR and sequencing techniques were carried out to investigate the underlying mechanism of resistance.RESULTS:E. coli IFI-1 and K. pneumoniae IFI-2 exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. Notably, both strains had up to a 32-fold higher level of resistance to ceftazidime than to cefotaxime. Further characterization revealed that a novel bla(CTX-M) gene encoding a beta-lactamase with a pI of 8.9 was implicated in this resistance: CTX-M-23. Along with the substitutions D114N and S140A, CTX-M-23 differed from CTX-M-1, the most closely related enzyme, by a P167T replacement in the active-site omega loop, which has not previously been observed in other CTX-M enzymes. By analogy with what was observed with certain TEM/PSE/BPS-type beta-lactamases, the amino acid substitution in the omega loop may explain ceftazidime resistance, which has only rarely been reported for other CTX-M enzymes.CONCLUSION:The emergence of a new ceftazidime-resistant CTX-M-type mutant provides evidence that these enzymes are able to broaden their substrate spectrum towards ceftazidime, probably due to substitutions in the active-site omega loop.

OBJECTIVES:In this study, we evaluated the performance of a new ESBL Etest configuration based on clavulanate synergy with cefepime compared with cefotaxime-clavulanate and ceftazidime-clavulanate ESBL Etest strips for the detection of extended-spectrum beta-lactamases (ESBL) in an Enterobacteriaceae strain collection, with special focus on Enterobacter spp.METHODS:Overall, a total of 54 clinical isolates of ESBL-producing Enterobacteriaceae species were evaluated: Enterobacter aerogenes (n=3), Enterobacter cloacae (n=10), Escherichia coli (n=10), Klebsiella oxytoca (n=3), Klebsiella pneumoniae (n=25) and Proteus mirabilis (n=3). To check Etest behaviour with resistance phenotypes similar to ESBL, our panel was expanded by six clinical isolates of K. oxytoca that were identified as putative producers of their chromosomal K1 beta-lactamase.RESULTS:With this panel, ESBL Etest was 98% sensitive with cefepime-clavulanate, 83% with cefotaxime-clavulanate, and 74% with ceftazidime-clavulanate strips. Concentrating on Enterobacter spp., reliable ESBL detection could only be achieved by the new cefepime-clavulanate strip since it confirmed ESBL production in all strains (100% sensitivity) whereas only 4/13 (31%) of Enterobacter strains were positive using cefotaxime-clavulanate or ceftazidime-clavulanate strips. A limitation of using the new cefepime strip was less than optimal specificity with K1 phenotypes of K. oxytoca: among six strains, four isolates were scored false-positive by Etest strips containing cefepime-clavulanate.CONCLUSION:The new Etest ESBL strip containing cefepime-clavulanate is a valuable supplement to current methods for detection of ESBLs. In our study collection, the cefepime-clavulanate strip was the best configuration for detection of ESBLs, particularly in Enterobacter spp.

Wir untersuchten 165 Muttermilchproben auf dasHepatitis-C-Virus (HCV). Sie wurden von 143 chronischHCV-infizierten Müttern gewonnen, die bei 152 Entbindungen 153Kinder zur Welt brachten. Die Entnahme erfolgte zwischen dem 31.Tag vor und dem 384. Tag nach der Entbindung (Median: 4 Tagepost partum). Keine der 165 Milchproben enthielt HCV-RNA. Wirzeigten, dass Bestandteile der Milch keine inhibitorischeWirkung auf den Nachweis des HCV mittels PCR haben. Dazu wurden120 Proben mit jeweils einem Serum eines HCV-positiven und einesHCV-negativen Patienten versetzt. In den mit virushaltigem Serumversetzten Milchproben konnte in 79 von 99 Fällen ein positivesPCR-Ergebnis gefunden werden. Von 134 Müttern stand eininnerhalb der Schwangerschaft oder bis zum 6. Monat danach(Median: 3 Tage post partum) gewonnenes Serum zur Bestimmung desVirustiters zur Verfügung. Neunundachtzig Frauen (66,4%)erwiesen sich als virämisch, die Viruskonzentrationen lagenzwischen 1×102 und1×107 Kopien/ml(Median:6×103 Kopien/ ml). Von den153 gestillten Kindern konnten wir 151 auf eine HCV-Infektionuntersuchen. Dabei erwiesen sich 3 Kinder (2,0%) als infiziert.Diese Übertragungsrate ist niedriger als die von 4,0% beiunserem bisher untersuchten Gesamtkollektiv von 624HCVexponierten Kindern. Zwei der 3 HCV-positiven Kinder wurdenbereits in utero infiziert, bei dem dritten Kind wurde das Virusspät in der Schwangerschaft oder unter der Geburt übertragen.Diese Befunde zeigen, dass die chronische HCV-Infektion einerFrau keine Kontraindikation für das Stillen darstellt.

BACKGROUND:In a HCV genotype 3a-infected patient, viremia with a different genotype (1b) was detected after 16 weeks of ineffective therapy. Serological typing revealed that this genotype had already been present prior to therapy.OBJECTIVES:To investigate the epidemiology of multiple HCV infections and the therapeutical consequences for patients superinfected with a new HCV strain.METHODS:Sera of 600 patients were screened for infection with multiple genotypes by using sequencing and a serological assay in parallel.RESULTS:Infection with two different HCV types was detected in 13 patients. The prevailing strain was genotyped by sequencing. From two of these patients additional sera were available which had been drawn up to 24 and 28 months prior to the current sample, respectively. Those early samples showed viremia with a HCV subtype that could not be detected by PCR afterwards. Only antibodies to the initial strain were detectable in the later samples.CONCLUSION:In patients serially infected by different HCV strains, one strain will prevail as the viremic virus. Under antiviral therapy, the displaced strain may become viremic again and may influence the outcome of therapy. Detection of inferior strains by serological assays before antiviral therapy may be important for choosing the adequate regimen.

The present study compares the ability to detect extended-spectrum β-lactamases (ESBL) among a collection of 34 ESBL producing clinical isolates belonging to Escherichia coli and Klebsiella species with two new rapid susceptibility and identification instruments—VITEK2 (bioMérieux, Marcy l’Etoile, France) vs. BDPhoenix (BD Biosciences, Sparks, MD). ESBL content in these isolates was previously characterized on the basis of PCR amplification and sequencing results which were used as the reference method in our evaluation. BDPhoenix correctly determined the ESBL outcome for all strains tested (100% detection rate), whereas VITEK2 was not able to detect the ESBL status in 5 isolates (85% detection rate). Detailed analysis revealed that the discrepancies were mainly observed with ‘difficult-to-detect’ strains. Misidentification was either due to low oximino cephalosporin MIC in these strains or was associated with pronounced ‘cefotaximase’ or ‘ceftazidimase’ phenotypes. Klebsiella oxytoca chromosomal β-lactamase (K1) is phenotypically quite similar to ESBL enzymes. In order to evaluate whether the K1 and ESBL enzymes could be discriminated, we expanded our analysis by 8 clinical K. oxytoca strains with K1 phenotypes. VITEK2 gave excellent identification of these strains whereas 7 out of 8 were falsely labeled ESBL-positive by the BDPhoenix system.

BACKGROUND:A recently discovered DNA virus (SEN) has been assumed to be responsible for posttransfusion hepatitis in humans. Phylogenetic analysis of SEN virus has revealed the existence of 8 different strains. Two of them (SEN virus strain H (SENV-H) and SENV-D) have been described as possible candidate viruses for inducing posttransfusion hepatitis. Until now, it is unclear whether patients on maintenance hemodialysis are on increased risk for acquiring SEN virus.OBJECTIVES:To investigate the prevalence of SENV-H among patients on maintenance hemodialysis and to examine whether special measures have to be taken to prevent nosocomial spreading of the virus.STUDY DESIGN:Serum samples derived from 78 chronically hemodialysed patients were examined for SENV-H viremia by seminested polymerase chain reaction. A panel of 226 samples from healthy blood donors served as a control group.RESULTS:The prevalence of SENV-H was determined to be 12.8% (n=10) among patients on maintenance hemodialysis. This is nearly the same prevalence as in healthy blood donors (16.8%; n=38). None of the solely SENV-H-viremic individuals had clinical or biochemical signs of liver disease. Enhanced severity of liver disease could not be observed in patients coinfected with hepatitis C virus and SENV-H.CONCLUSION:We conclude that SENV-H viremia is widespread among hemodialysis patients. Since no viremic patient had clinical or biochemical signs of liver disease, in our setting the hepatitis-inducing capacity of SENV-H remains unclear. On the basis of our results, at present, we do not regard it as necessary to dialyse SENV-H-viremic patients on separate machines.

Saliva antibodies to Escherichia coli O157 were investigated as markers of the immune response in children with enteropathic hemolytic uremic syndrome (HUS). Paired serum and saliva samples were collected from 22 children with HUS during acute disease and convalescence and were tested for E. coli O157 lipopolysaccharide (LPS)-specific IgM and IgA antibodies by ELISA. Serum and saliva samples from 44 age-matched controls were used to establish the cut-off values. Elevated levels of IgM and/or IgA antibodies to O157 LPS were detected in saliva of 13/13 HUS patients with Shiga toxin-producing E. coli (STEC) O157 in stool culture and from 4 of 5 HUS patients in whom STEC were not detected. These results closely mirrored the results obtained with paired serum samples. In contrast, saliva and serum samples from four children with STEC isolates belonging to O-groups O26, O145 (n = 2), and O165 lacked detectable O157 LPS-specific antibodies. The specificity of the ELISA was confirmed by western blotting. In STEC O157 culture-confirmed cases, the sensitivity of the ELISA was 92% for saliva IgM and IgA, based on the first available sample, and 100% and 92%, respectively, when subsequent samples were included. The specificity was 98% for IgM and 100% for IgA. Children with E. coli O157 HUS demonstrate a brisk, easily detectable immune response as reflected by the presence of specific antibodies in their saliva. Saliva-based immunoassays offer a reliable, noninvasive method for the diagnosis of E. coli O157 infection in patients with enteropathic HUS.