A randomized trial in patients undergoing percutaneous coronary angioplasty: roxithromycin does not reduce clinical restenosis but angioplasty increases antibody concentrations against Chlamydia pneumoniae.

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.

A new combined HIV p24 antigen and anti-HIV-1/2/O screening assay.

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.

Prolonged time until seroconversion among hemodialysis patients: the need for HCV PCR.

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.

Evaluation of a new screen agar plate for detection and presumptive identification of Enterobacteriaceae producing extended-spectrum beta-lactamases.

A new agar screen plate for extended-spectrum beta-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.

Disseminated Mycobacterium haemophilum infection as initial manifestation of AIDS.

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.

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria.

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.

A novel extended-spectrum beta-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance.

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.

Evaluation of a new cefepime-clavulanate ESBL Etest to detect extended-spectrum beta-lactamases in an Enterobacteriaceae strain collection.

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.

Pet snakes as a reservoir for Salmonella enterica subsp. diarizonae (Serogroup IIIb): a prospective study.

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.

Viral features of lamivudine resistant hepatitis B genotypes A and D.

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 204I 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 rt204I 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 rt204I 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.

Multiple infections with different HCV genotypes: prevalence and clinical impact.

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.

A novel DNA virus (SEN) among patients on maintenance hemodialysis: prevalence and clinical importance.

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.

Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum beta-lactamase detection in Escherichia coli and Klebsiella species clinical isolates.

The present study compares the ability to detect extended-spectrum beta-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 beta-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.

In vitro activity of moxifloxacin against bacteria isolated from odontogenic abscesses.

We evaluated the antimicrobial susceptibility of 87 pathogens isolated from 37 patients with odontogenic abscesses. The most prevalent bacteria were viridans group streptococci and Prevotella species. Considering all bacterial isolates, 100% were susceptible to amoxicillin-clavulanic acid, 98% were susceptible to moxifloxacin and to levofloxacin, 76% were susceptible to doxycycline, 75% were susceptible to clindamycin, and 69% were susceptible to penicillin.

Saliva IgM and IgA are a sensitive indicator of the humoral immune response to Escherichia coli O157 lipopolysaccharide in children with enteropathic hemolytic uremic syndrome.

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.

In vitro activity of polyoxin D and nikkomycin Z against Encephalitozoon cuniculi.

Microsporidia of the genus Encephalitozoon are emerging protozoal agents that mainly infect immunocompromised patients with AIDS. At present, disseminated infections with members of the genus Encephalitozoon can only be successfully treated with albendazole. As chitin is a basic component of the microsporidian spore. we evaluated, in vitro, the susceptibility of a human-derived strain of Encephalitozoon cuniculi to polyoxin D and nikkomycin Z, which are known competitive inhibitors of chitin synthetase enzymes. Using an in vitro assay, polyoxin D at 1, 10 and 100 microg/ml significantly reduced the number of parasitic foci on days 6, 9, and 15 post-infection. However, nikkomycin Z revealed a marked but lower reduction in the number of parasitic foci than polyoxin D. A significant reduction of parasitic foci was achieved for nikkomycin Z at 10 and 100 microg/ml up to day 9 post-infection. Polyoxin D was approximately tenfold more effective in our in vitro assay than nikkomycin Z.

Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome.

Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.

Epidemiological dynamics of hepatitis C virus among 747 German individuals: new subtypes on the advance.

This study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.