Bed rails and endotracheal tube connectors as possible sources for spreading Acinetobacter baumannii in ventilator-associated pneumonia patients.

This study aimed to determine molecular patterns of Acinetobacter baumannii using a PCR-based technique with REP-1, REP-2 and M13 primers to distinguish the patients' strains and the environmental strains (condensate, endotracheal tube connector, bed rail and nurses hands). There were 67 cases of ventilator-associated pneumonia (VAP) among 600 patients using mechanical ventilators in 10 wards from March to July 2006. The incidence of VAP was 11.2% or 8.9/1,000 ventilator days with a 54.5% fatality rate. Among 19 of 22 A. baumannii VAP patients, 68.4% (13/19) had their environmental samples contaminated with A. baumannii and the most common contaminated sites were bed rails and endotracheal tube connectors (36.8% each). Multidrug resistant (MDR) A. baumannii were involved in 77.3% of A. baumannii VAP. Molecular typing of 96 A. baumannii isolates was able to differentiate A. baumannii isolates into 7 types. Type 2 was the most common and found in 77.3% (17/22) of A. baumannii VAP patients admitted in 6 of 7 wards. Identical fingerprints were found in clinical isolates and their bed rails, endotracheal tube connectors and condensates of 5 patients. The results demonstrate that multiple clones of MDR A. baumannii were widely spread in the hospital. Bed rails and contaminated endotracheal tube connectors could be potential sources of A. baumannii spread.

Cockroach allergen detection and cockroach allergens of allergic Thai patients.

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.

Recombinant American cockroach component, Per a 1, reactive to IgE of allergic Thai patients.

Twelve similar recombinant Per a 1 clones were produced from an American cockroach (CR) cDNA library. The nucleotide sequence of a representative cline, i.e. clone A6, contained 579 base pairs (bp) and a 372 bp open reading frame (2-373) encoding 124 amino acids. A stop codon was found at position 374-376 followed by a 3' end untranslated region with an AATAAA polyadenylation signal and a poly (A) tail. The estimated molecular mass of the 24 amino acid residue protein was 13.8 kDa, with a predicted isoelectric point value of 4.74. Cysteine or N-linked glycosylation was not found. The deduced amino acid sequence of the A6 revealed 84.68-95.97% identity to other previously reported Per a 1 clones and 65.87-69.60% homology to the previously reported Bla g 1 clones. However, while previously reported Per a 1 clones showed homology to ANG12, a precursor protein in the midgut of the female Anopheles gambiae secreted after the blood meal, the A6 DNA sequence was found to have homology (37.1%) to DNA of G2, a putative protein in the midgut of Aedes aegypti (AY 050565). The deduced amino acid sequence of A6 contained a mitochondrial energy transfer protein signature, phosphorylation sites for the cAMP-and cGMP-dependent protein kinase C and casein kinase II. Hydrophobic and hydrophilic characteristics of the A6 deduced peptide indicated that it was a transmembrane protein. This is the first report that Per a 1 is a transmembrane protein. The deduced amino acid sequence of the A6, which contained the sequence LIRSLFGLP, differed in one amino acid from two previously reported epitopes, i.e. LIRALFGL and IRSWFGLP, of Per a 1.0104 which bound 80% and 100%, respectively, to IgE of the allergic patients tested. The A6 DNA sequence was deposited in the GenBank (Accession number AY 259514) and has been designated Per a 1.0105. The A6 expressed protein bound to monoclonal antibodies (MAb 3C2) specific to American cockroach and also bound to IgE of all (100%) of the 20 allergic Thai patients.