Molecular typing of multidrug-resistant Salmonella Blockley outbreak isolates from Greece.

During 1998, a marked increase (35 cases) in human gastroenteritis due to Salmonella Blockley, a serotype rarely isolated from humans in the Western Hemisphere, was noted in Greece. The two dominant multidrug-resistance phenotypes (23 of the 29 isolates studied) were associated with two distinct DNA fingerprints, obtained by pulsed-field gel electrophoresis of genomic DNA

Antibiotic sensitivity profile of Salmonella isolated from two slaughterhouses and human clinical cases

The antibiotic sensitivity of Salmonella strains isolated during the period 1996-98 from two industrial slaughterhouses of Northern Greece was detennined and compared with that of salmonellae isolated from human hospital cases during the period 1995-1997. For antibiotic sensitivity the disc agar diffusion method was used. Of 1874 samples obtained from the slaughterhouse environment (floors, worker\u27s hands and their knives), pork carcasses, by-products (livers and plucks) as well as lymph nodes and caecal contents 178 (9.5%) were positive for Salmonella spp. The salmonellae belonged to 22 serotypes. S. derby, S. london and S. typhimurium represented 25.8%, 15.2%, and 10.7% of the serotypes respectively. Of the salmonellae 59%, and 4.5%, were resistant and 33%, and 4.5% were intermediate sensitive to Tetracyclin, and Streptomycin, respectively and 26.4%, 14.6%, 5.1%, 1.7% and 1% were resistant to Ampicillin, Sulfamethoxa\u3eole I Trimethoprim, Chloramphenicol, Gentamicin, and Tobramycin respectively. Of the S. typhimurium strains 47% were resistant to Ampicillin and 41.2% to Chloramphenicol. Seven of the 19 strains were DT I 04, isolated for the first time in Greece, and multiple drug resistant. Of all isolates 5.1% were resistant to Chloramphenicol, the use of which is prohibited in food animal veterinary practice. Of the 422 salmonellae isolated at the Hospital of Infectious Diseases in Thessaloniki during the period 1996-98 77.4% were S. enteritidis and 17.7% S. typhimurium. Of the salmonellae isolated during 1995-1997, 76-79 % were resistant to Ampicillin and 1.2-1.5% to Chloramphenicol. Many of S. typhimurium strains isolated from the slaughterhouses and human cases exhibited the same antibiotic sensitivity profile a fact indicative of a potential transfer of animal strains to humans. Salmonellae of the same serotype exhibited different antibiotic resistance profiles, an indication of the presence of different clones within the same serotype. No S. enteritidis was isolated in slaughterhouses

Antibiotic sensitivity profile of Salmonella isolated from two slaughterhouses and human clinical cases

The antibiotic sensitivity of Salmonella strains isolated during the period 1996-98 from two industrial slaughterhouses of Northern Greece was detennined and compared with that of salmonellae isolated from human hospital cases during the period 1995-1997. For antibiotic sensitivity the disc agar diffusion method was used. Of 1874 samples obtained from the slaughterhouse environment (floors, worker's hands and their knives), pork carcasses, by-products (livers and plucks) as well as lymph nodes and caecal contents 178 (9.5%) were positive for Salmonella spp. The salmonellae belonged to 22 serotypes. S. derby, S. london and S. typhimurium represented 25.8%, 15.2%, and 10.7% of the serotypes respectively. Of the salmonellae 59%, and 4.5%, were resistant and 33%, and 4.5% were intermediate sensitive to Tetracyclin, and Streptomycin, respectively and 26.4%, 14.6%, 5.1%, 1.7% and 1% were resistant to Ampicillin, Sulfamethoxa>ole I Trimethoprim, Chloramphenicol, Gentamicin, and Tobramycin respectively. Of the S. typhimurium strains 47% were resistant to Ampicillin and 41.2% to Chloramphenicol. Seven of the 19 strains were DT I 04, isolated for the first time in Greece, and multiple drug resistant. Of all isolates 5.1% were resistant to Chloramphenicol, the use of which is prohibited in food animal veterinary practice. Of the 422 salmonellae isolated at the Hospital of Infectious Diseases in Thessaloniki during the period 1996-98 77.4% were S. enteritidis and 17.7% S. typhimurium. Of the salmonellae isolated during 1995-1997, 76-79 % were resistant to Ampicillin and 1.2-1.5% to Chloramphenicol. Many of S. typhimurium strains isolated from the slaughterhouses and human cases exhibited the same antibiotic sensitivity profile a fact indicative of a potential transfer of animal strains to humans. Salmonellae of the same serotype exhibited different antibiotic resistance profiles, an indication of the presence of different clones within the same serotype. No S. enteritidis was isolated in slaughterhouses.</p

Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR

Human brucellosis poses a significant public health problem in many developing countries and requires fast and accurate diagnosis. A PCR assay amplifying part of the 31-kDa Brucella abortus antigenic protein gene sequence was developed and applied to whole-blood and serum samples from 31 brucellosis patients and 45 healthy individuals. All patients except one had detectable Brucella DNA in either whole blood or serum (combined sensitivity, 97%), but, the assay sensitivity was higher with serum samples (94%) than with whole-blood samples (61%). The assay specificity was excellent (100%). A confirmatory PCR assay targeting another Brucella gene region (omp-2) was also developed but lacked sensitivity. Serum is the optimal specimen for the diagnosis of brucellosis by PCR, a choice that leads to assay simplification and shortens turnaround time

Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers amplifying C. neoformans URA5 gene sequences. To test compatibility of different DNA extraction protocols with the PCR-restriction fragment length polymorphism (RFLP) assay, a commercial DNA extraction kit (XTRAX™; Gull Laboratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted DNA from spiked clinical samples containing C. neoformans (8±2 cells ml−1) and generated amplification products suitable for restriction enzyme analysis. Alu I digestion differentiated the two varieties of C. neoformans. Three distinct RFLP patterns were obtained upon restriction with MspI corresponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analysis, coupled with a sensitive in-house or commercially available DNA extraction method from clinical samples, could be successfully incorporated into rapid routine diagnostic strategies. It could also provide an expeditious tool for epidemiology-based population genetics studies

Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers amplifying C. neoformans URA5 gene sequences. To test compatibility of different DNA extraction protocols with the PCR-restriction fragment length polymorphism (RFLP) assay, a commercial DNA extraction kit (XTRAX(TM); Gull Laboratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted DNA from spiked clinical samples containing C neoformans (8 +/-2 cells ml(-1)) and generated amplification products suitable for restriction enzyme analysis. Alu I digestion differentiated the two varieties of C neoformans. Three distinct R-FLP patterns were obtained upon restriction with MspI corresponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analysis, coupled with a sensitive in-house or commercially available DNA extraction method from clinical samples, could be successfully incorporated into rapid routine diagnostic strategies. It could also provide an expeditious tool for epidemiology-based population genetics studies

Evaluation and comparison of fluorescence polarization assay with three of the currently used serological tests in diagnosis of human brucellosis

Fluorescence polarization assay (FPA) is a method that has been used for the diagnosis of brucellosis in animals for many years. To test its possible usefulness for the diagnosis of human brucellosis, 230 sera from patients with clinical signs of brucellosis and positive serological tests (Rose Bengal, Standard Agglutination Test, iELISA), and 305 sera from a healthy population with no clinical/epidemiological/serological evidence were examined with FPA. By using ROC analysis, the cut-off value was estimated at 99 mP, with 93.5% sensitivity (95% CI 89.5-96.3) and 96.1% specificity (95% CI 93.2-97.9). The pairwise comparison of ROC curves between FPA and iELISA and between FPA and RBT revealed no significant statistic difference (P 0.05). SAT also had the lowest sensitivity (81.7%) among the three tests used in case definition while iELISA had a sensitivity of 90.8% and RBT a sensitivity of 88.7%. The Kappa analysis showed that FPA has a very good agreement (0.92) with the "status of the disease" and with iELISA (0.837). According to our results, FPA seems to be a valuable method for the diagnosis of brucellosis in humans. Taking into consideration the advantages of the method such as the speed of results obtaining, the objectivity of results interpretation, as well as the cost, FPA could be considered as a replacement for other established methods. However, further studies are needed to assess the reproducibility of FPA

Repeated Occurrence of Diverse Extended-Spectrum β-Lactamases in Minor Serotypes of Food-Borne Salmonella enterica subsp. enterica

Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum β-lactamase (ESBL)-producing human isolates. Salmonella enterica serotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. enterica serotype Blockley and S. enterica serotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. enterica serotype Virchow strain producing plasmid-mediated CTX-M-32 was isolated twice from poultry end products. All ESBL plasmids were self-transferable and carried by clones currently common in Greece

Phenotypic assessment of Neisseria meningitidis isolates obtained from patients with invasive meningococcal disease in Greece, 1993-2003: Implications for serogroup B vaccines based on PorA serosubtype antigens

Serogroup B is the major isolate from patients with invasive meningococcal disease (IMD) in Greece. This study used the whole cell enzyme-linked immuosorbent assay (ELISA) with monoclonal antibodies to screen Neisseria meningitidis isolates obtained from patients with IMD between 1993 and 2003 to determine if serosubtypes included in the hexavalent Por A OMP vaccines being tested in northern Europe were prevalent in Greece. During this period there were significant changes in the proportions of serogroups B and C isolated from patients. Serogroup C was predominant in 1996-1997 but fell sharply with corresponding increases in serogroup B. Of the 591 isolates sent to the National Meningitis Reference Laboratory in Athens during this period, 325 (55%) were serogroup B. Among those tested for serosubtype, porA proteins used for the vaccine being tested in Britain were detected on 85/284 (30%) strains and for the vaccine being tested in the Netherlands 175/284 (62%). P1.14 (58/284, 20%) the predominant serosubtype among the Greek isolates, is not present in either vaccine formulation; 23/284 (8%) strains did not react with any of the monoclonal antibodies. Our results indicate that introduction of the vaccines currently being evaluated in northern Europe would not be warranted in the Greek population. © 2005 Elsevier Ltd. All rights reserved