Prevalence of thrombophilia in asymptomatic individuals with a family history of thrombosis.

OBJECTIVE: The present study investigates the prevalence of thrombophilia in individuals with first or/and second degree family history of thromboembolism. MATERIAL-METHODS: The study group consisted of 68 individuals with a first or second degree family history of venous or arterial thromboembolism, but without a personal history of thrombosis. The activity of ATIII, PC, PS, FVIII, FΧΙΙ and total homocysteine was measured on the ACL Advance coagulation analyzer. In addition, hemi-quantitative determination of CRP was performed to exclude an acute phase reaction. The existence of V-Leiden mutation was investigated by the modified pre-dilution method (1:5) with V-DEF. Prothrombin G20210A mutation was detected by the use of an in house PCR protocol. Family history was termed as follows: positive (thrombosis was reported in one parent and his/her family members) (group Α) or strongly positive (thrombosis was reported in both parents and their family members (group Β). RESULTS: Data analysis revealed decreased activity of ATIII:1.47%, PC:1.47%, and FXII:5.9%, increased activity of FVIII (without inflammation):11.8%, V-Leiden:13%, elevated Hcy:14.7%, and G20210A mutation:2.9%. Group A consisted of 55 individuals (74.5%), whereas group B of 16 (23.5%). The total percentage of thrombophilia was 48.5%, while the percentage within group A was 44.2% and within group B 62.5%. CONCLUSION: The high prevalence of thrombophilia, reinforce the importance of an extensive laboratory thrombophilia screening when a family history of thromboembolism has been recorded, especially when it concerns both parents and/or their family members and even more when one or more acquired thrombophilic factors coexist.

Typing of Legionella pneumophila strains isolated in Greece by arbitrarily-primed PCR.

Genomic fingerprints from DNA of fifteen environmental and clinical strains of Legionella pneumophila serogroup 1, isolated from diverse geographic areas of Greece, during the period 1986 to 1994, were generated with arbitrarily-primed polymerase chain reaction (AP-PCR) in order to use the discriminatory power of two arbitrary primers, BG2 and M13 Forward to clarify the relationship among the fifteen isolates. Both primers were found to have the ability to discriminate strains of the same serogroup, to identify strains related to each other even though they were isolated at different times. Therefore, AP-PCR using BG2 or M13 Forward as primers, appears to be a useful tool which provides a fast and simple method for epidemiological fingerprinting.

Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples.

OBJECTIVE: To apply the polymerase chain reaction (PCR) to serum samples for the rapid diagnosis of Legionnaire's disease using the L5SL9 and L5SR93 primers designed to generate a 104-base-pair (bp) fragment from the 5S RNA gene of Legionella spp. The amplified product was detected by electrophoresis and by hybridization with the L5S-1-specific probe. METHODS: Single specimens of serum obtained from 24 patients with confirmed legionellosis, at different stages of their disease, were tested by PCR. Additionally, 10 serum samples from patients with no clinical symptoms of pneumonia and 10 samples from patients suffering from pneumonia caused by Mycoplasma pneumoniae, Coxiella burnetii or Chlamydia psittaci were also tested as controls in order to determine the specificity of the method. RESULTS: Of the 24 examined serum samples, the amplified products from 12 hybridized with the L5S-1 probe (sensitivity 50%). None of the negative controls was positive after PCR. No correlation was found between the day of illness and the positivity in the test. CONCLUSIONS: The PCR technique could be applied as a diagnostic tool for the rapid diagnosis of legionellosis in serum samples after modification, mainly to improve its sensitivity.

Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis.

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20-1.00 and epidemiological concordance (E) values of 0.11-1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78-1.00, and E=0.67-1.00, with 10-20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing Using a standard protocol.

The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i). reports recommendations for the designation of the European Working Group on Legionella Infections (EWGLI) AFLP types, (ii). describes the EWGLI AFLP types identified for the 130 strains in the EWGLI culture collection, and (iii). reports the results of a newly introduced international programme of proficiency testing. Following preliminary analysis of 20 epidemiologically unrelated isolates, 16 major AFLP types were identified. A coded proficiency panel, comprising 12 additional isolates representing 9 of these 16 AFLP types, was sent to 17 centres in 14 European countries where it was analysed following a previously determined standard protocol. The identity of each coded strain (recorded as AFLP type 001-016 or untypeable) was determined by participants with reference to these 16 AFLP types, either visually or using gel analysis software where available, and reported to the coordinating centre. Nine of the 12 strains, including an epidemiologically related pair and two pairs of unrelated isolates of the same type, were correctly identified to the correct AFLP type by all or all but one of the participants. Seven laboratories correctly identified all 12 isolates, and a further seven laboratories correctly identified 11. Type identification scores ranged from 75% (1 centre), 83% (2 centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied.