Search PubMed⌕ Search

Biomedical subjects

N J Legakis

Publications and source records attributed to N J Legakis.

At least 55 records · Page 3Linked to original sources
PubMed2001

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.

Adult↗
PubMed2000

CTX-M-type beta-lactamases: an emerging group of extended-spectrum enzymes.

CTX-M-type beta-lactamases constitute a novel group of class A beta-lactamases with extended-spectrum properties. They are encoded by transferable plasmids and found in various enterobacteria, mostly Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. CTX-M enzymes share extensive sequence similarity with the chromosomal beta-lactamases of Klebsiella oxytoca. They efficiently hydrolyze many newer broad-spectrum oximino-beta-lactams including cefotaxime, ceftriaxone and aztreonam and are readily inhibited by tazobactam and clavulanate. CTX-M-producing enterobacteria are endemic in Latin America and in some areas of North Eastern Europe. Data on their structure, properties and epidemiology are discussed.

Amino Acid Sequence↗
PubMed2000

Pneumococcal acute otitis media in children.

OBJECTIVES: To evaluate the clinical and laboratory findings of Streptococcus pneumoniae acute otitis media in children during a 1 year period. METHODS: From October 1995 to September 1996, 113 children aged 2 months to 14 years (median 18 months), with S. pneumoniae acute otitis media were studied. Susceptibility testing was performed by the Kirby-Bauer method and the E-test, and serotyping by the Quellung reaction. RESULTS: E-test assays detected five isolates (4.4%) to be highly resistant to penicillin and 13 (11.5%) that had intermediate resistance. All isolates were found to be susceptible to vancomycin, rifampicin and cefotaxime. In total, 25 isolates (22.1%) were resistant to one or more drugs. Fifty per cent of the penicillin-resistant or intermediately resistant S. pneumoniae isolates were resistant to multiple drugs, whereas only 2.1% of the penicillin-susceptible isolates were resistant to multiple drugs. The predominating serogroups of the isolates with reduced susceptibility to penicillin were the 19 (61.1%), 9 (16.7%), 23 (11.1%), 6 (5.5%) and 14 (5.5%) whereas those of the susceptible isolates were the 19 (26.3%), 14 (13.7%), 3 (11.6%), 6 (11.6%), 9 (8.4%), 1 (5.3%) and 12 (5.3%). CONCLUSIONS: Streptococcus pneumoniae isolates from children with acute otitis media were penicillin-insensitive in 15.9%. The multiresistant S. pneumoniae isolates belonged to serogroups: 19 (45.4%), 9 (27.3%), 6 (18.2%) and 23 (9.1%).

Acute Disease↗
PubMed2000

Comparison of Mycobacterium avium isolates from Greek AIDS and human immunodeficiency virus-negative patients by pulsed-field gel electrophoresis.

OBJECTIVE: To compare the chromosomal types of Mycobacterium avium strains infecting HIV-negative and AIDS patients in Greece. METHODS: In total, 41 Mycobacterium avium isolates, 23 from AIDS and 18 from HIV-negative patients, were compared by pulsed-field gel electrophoresis of genomic DNA after XbaI digestion. The majority (87%) of AIDS isolates were from disseminated infection, while the majority (61%) of HIV-negative isolates were from children with cervical lymphadenitis. RESULTS: Pulsed-field gel electrophoresis classified strains whose electrophoretic patterns were at least 85% similar into three clusters, A (four isolates), B (12 isolates), and C (15), while 10 isolates remained outside of these clusters. There was no statistically significant correlation of any PFGE cluster with a specific patient group. Within each patient group, no significant correlation of PFGE type with time, place of residence or, in the case of AIDS patients, hospital attended was observed. CONCLUSIONS: Genotypic similarities between isolates responsible for disseminated infection in AIDS patients and lymphadenitis in HIV-negative children suggest that related strains, possibly from an environmental source, cause both types of infections.

AIDS-Related Opportunistic Infections↗
PubMed2000

Detrimental effect of the combination of R164S with G238S in TEM-1 beta-lactamase on the extended-spectrum activity conferred by each single mutation.

The non-naturally occurring TEM-1 beta-lactamase mutant R164S:G238S, as well as the R164S (TEM-12) and G238S (TEM-19) beta-lactamases, were constructed and expressed in Escherichia coli under isogenic conditions. Comparison of susceptibilities to beta-lactam antibiotics and substrate profiles showed that the combination of R164S with G238S drastically reduced the extended-spectrum activity of the respective single mutants.

Amino Acid Substitution↗
PubMed2000

Diversity among high-level aminoglycoside-resistant enterococci.

A total of 55 Enterococcus faecalis and 21 Enterococcus faecium non-replicate isolates were obtained from routine clinical specimens, during a 1 year period, in a tertiary care hospital in Athens, Greece. The most common isolation site was the urinary tract (44% of E. faecalis and 33% of E. faecium isolates). No vancomycin resistance was detected. Ampicillin-resistant isolates did not produce beta-lactamase. High-level gentamicin resistance was detected in 22% and 0% of E. faecalis and E. faecium isolates, respectively. The corresponding figures for high-level streptomycin resistance were 40% and 33%. The aminoglycoside-modifying enzyme gene aac(6')+aph(2") was detected by PCR in 10 of 12 high-level gentamicin-resistant E. faecalis isolates, and the ant(6)-I gene in all high-level streptomycin-resistant isolates of both species. DNA fingerprinting by PFGE grouped 31 of 55 E. faecalis isolates into 10 clusters, and 10 of 21 E. faecium isolates into two clusters, containing two to seven isolates each. Two E. faecalis PFGE types, comprising isolates expressing high-level aminoglycoside resistance, and not observed among non-high-level aminoglycoside-resistant strains, were disseminated in building A of the hospital. In contrast, high-level aminoglycoside resistance seemed to have been acquired nosocomially by a number of genotypically different E. faecium types. Molecular typing was therefore instrumental in understanding the differences in the mode of spread and acquisition of high-level aminoglycoside resistance among these two different enterococcal species.

Aminoglycosides↗
PubMed2000

Multicenter evaluation of epidemiological typing of methicillin-resistant Staphylococcus aureus strains by repetitive-element PCR analysis. The European Study Group on Epidemiological Markers of the ESCMID.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Bacterial Typing Techniques↗
PubMed2000

Multiple clones within multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104. The Greek Nontyphoidal Salmonella Study Group.

Six distinct clones were present among Greek multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3'')-Ia, pse-1, and tem-1.

Animals↗
PubMed1999

Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples.

In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.

Aspartic Acid Endopeptidases↗
PubMed1999

Gradual increase in the minimum inhibitory concentration of penicillin among both susceptible and resistant Streptococcus pneumoniae isolates from Greek children during 1995-1997.

A total of 140 non-replicate Streptococcus pneumoniae community isolates from Greek children collected during the period 1995-1997 were studied. Combined intermediate and high penicillin resistance rates were 23% in 1995, 29% in 1996, and 27% in 1997. The proportion of highly resistant isolates steadily increased from 2% in 1995 to 12% in 1997. There was no significant difference in penicillin resistance rates among colonizing and infecting isolates (23 and 27%, respectively). Over the study period, a clear shift towards higher penicillin MIC was observed among both the susceptible and resistant groups. Thus, penicillin resistance rates were equally high among colonizing and infecting isolates and resistance levels appeared to be gradually increasing throughout the entire S. pneumoniae population.

Child↗
PubMed1999

A multi-centre study of nosocomial methicillin-resistant Staphylococcus aureus in Greece. Greek MRSA Study Group.

All 105 non-replicate consecutive Staphylococcus aureus strains isolated in 1997 from seven Greek hospitals, were found to be susceptible to vancomycin, teicoplanin and chloramphenicol, but only five (8%) were susceptible to all 16 antibiotics tested. Forty-three (41%) isolates were methicillin-resistant, 58% homogeneously (homMRSA) and 42% heterogeneously (hetMRSA). Resistance of homMRSA strains to other antibiotics was generally high (88-100%), although only one strain was resistant to netilmicin. Resistance in hetMRSA (6-39%) or in MSSA (5-11%) was significantly lower. Consequently, the majority (76%) of homMRSA were multi-drug resistant, while the dominant phenotype of hetMRSA and MSSA was resistance to penicillin (50% and 76%, respectively). Comparison of these strains with isolates from 1994 showed higher resistance rates to erythromycin among MSSA, to erythromycin and amikacin among hetMRSA and to rifampicin among homMRSA strains.

Anti-Bacterial Agents↗
PubMed1999

Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 beta-lactamases.

In SHV-type beta-actamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 beta-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure. Wild-type SHV-1 and -5 beta-lactamases and their respective Asn276-->Asp mutants were expressed under isogenic conditions by cloning the respective bla genes into the pBCSK(+) plasmid and transforming Escherichia coli DH5alpha. Determination of IC50 showed that SHV-1(Asn276-->Asp), compared with SHV-1, was inhibited by 8- and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of Asn276 by Asp in SHV-5 beta-lactamase caused a ten-fold increase in the IC50 of clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and 5.5-fold, respectively. Beta-lactam susceptibility testing showed that both Asn276-->Asp mutant enzymes, compared with the parental beta-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino beta-lactams tested (cefotaxime, ceftriaxone and aztreonam). The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were slightly elevated in the clones producing the mutant beta-lactamases. The latter clones were also less susceptible to penicillin-inhibitor combinations. Asn276-->Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of TEM-1 beta-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed.

Amino Acid Substitution↗
PubMed1999

Reduced susceptibility to vancomycin of nosocomial isolates of methicillin-resistant Staphylococcus aureus.

The MICs of vancomycin for 56 random nosocomial Staphylococcus aureus isolates homogeneously resistant to methicillin (homMRSA), 16 heterogeneously resistant isolates (hetMRSA) and 25 susceptible isolates (MSSA) were determined by a standard broth microdilution method. Representative isolates were also tested by an agar incorporation method, the Etest and population analysis. Although always in the susceptible range, MICs of vancomycin for homMRSA were significantly higher than those for hetMRSA or MSSA. Moreover, a homMRSA strain belonging to one of the major Greek MRSA clones contained a sub-population of cells that could grow in the presence of vancomycin 8 mg/L at a frequency of 6.7 x 10(-8).

Anti-Bacterial Agents↗
PubMed1999

Identification of medically significant fungal genera by polymerase chain reaction followed by restriction enzyme analysis.

Rapid non-culture-dependent assays for identification of fungi quicken diagnosis and prompt treatment of invasive fungal disease. Fungal DNA extracts from pure cultures of the most frequently isolated fungal pathogens belonging to the Genera Aspergillus, Candida and Cryptococcus along with less common pathogenic Genera were amplified with the general fungal primer pair internal transcribed spacer-1/4. Subsequently, the amplicon was digested with the restriction endonucleases MspI, HaeIII, HinfI and EcoRI in order to generate genus- or species-specific patterns for identification of the fungus. HinfI produced indistinguishable fingerprints for all Aspergillus species tested. MspI produced species-specific patterns for: Cryptococcus neoformans, Cryptococcus non-neoformans, Candida albicans and Candida tropicalis. EcoRI succeeded in differentiating penicillia from aspergilli and cryptococci from Candida spp. It is concluded that this procedure can differentiate genera and occasionally species of medically important fungi and that following the necessary validation experiments, it can be used directly on clinical samples to assist prompt diagnosis of systemic fungal infections.

DNA Restriction Enzymes↗
PubMed1999

Oral lesions in children with perinatally acquired human immunodeficiency virus infection.

Fifteen vertically HIV-infected children aged between 2 and 12 years were followed up for 1 year, weekly to monthly, to study the incidence of oral lesions. At the time of first examination, oral candidiasis (OC) was observed in nine children. Seven children presented with the erythematous type only and two with pseudomembranous oral candidiasis. Four cases of cheilitis were seen in association with the erythematous forms of oral candidiasis. One erythematous candidiasis progressed to pseudomembranous form. A second case of erythematous OC, after multiple recurrences in the form of erythematous OC, recurred as pseudomembranous OC. Another case of erythematous OC and one of pseudomembranous OC presented after multiple recurrences as a persistent, adherent pseudomembranous OC. An orofacial herpes-zoster infection, a hairy leukoplakia and a necrotic lingual ulcer were observed as second lesions and in association with oral candidiasis in three children. Erythematous oral candidiasis was the most frequent oral HIV-related lesion, was observed in different stages of HIV-infection, and in some cases progressed to pseudomembranous candidiasis. A different, selectively resistant, Candida clone was isolated in three cases of recurrent candidiasis.

Antifungal Agents↗
PubMed1999

Paediatric AIDS--related linear gingival erythema: a form of erythematous candidiasis?

Three vertically HIV-infected children showed, in addition to oral candidiasis, HIV-gingivitis, which healed on antimycotic treatment. The intense linear gingival erythema of a fourth child was also clinically evaluated as a possible form of erythematous oral candidiasis. Direct microscopic examination of material from the gingival lesions of the latter disclosed yeast cells and hyphae. Subsequent culture, biochemical and serological tests identified the yeast as Candida dubliniensis. As the patient was on long-term prophylaxis with fluconazole, ketoconazole was administered and led to a good clinical response. This is the first report implicating this new Candida species as a pathogen in linear gingival erythema in a HIV-positive individual. The case reports presented provide evidence that linear gingival erythema may be of candidal origin. Further clinical and laboratory observations are required to establish whether this condition constitutes a variant of erythematous candidiasis associated with paediatric HIV infection.

Acquired Immunodeficiency Syndrome↗