- Volume 53, Issue 12, 2004

Chlamydophila pneumoniae, an obligately intracellular Gram-negative bacterium and a common causative agent of respiratory tract infections, has been implicated in the induction and progression of atherosclerosis and coronary artery disease. In this study, the signalling mechanism of C. pneumoniae in human fibroblasts, a prominent cell population in chronic inflammation and persistent infection, contributing to plaque formation, was investigated. C. pneumoniae elementary bodies were demonstrated to up-regulate the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) in human fibroblasts. The effect was independent of the chlamydial lipopolysaccharide and was likely to be mediated by a heat-labile chlamydial protein. Furthermore, an anti-Toll-like receptor 4 (TLR4) antibody was shown to abolish C. pneumoniae-induced cell activation, whereas an anti-TLR2 antibody had no effect, indicating the role of TLR4 in p44/p42 MAPK activation. Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 and phosphodiesterase 4 (PDE 4) inhibitor Rolipram enhanced C. pneumoniae-induced MAPK phosphorylation and attenuated C. pneumoniae infectivity in vitro. Together the results indicate that C. pneumoniae triggers rapid TLR4-mediated p44/p42 MAPK activation in human fibroblasts and chemical enhancement of MAPK phosphorylation modulates in vitro infection at the molecular level.

Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1–10 c.f.u. ml−1 of the sample. No amplification was observed from up to105 heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis.

Bartonella henselae is a recently recognized pathogenic bacterium associated with cat-scratch disease, bacillary angiomatosis and bacillary peliosis. A recombinant clone expressing an immunoreactive antigen of B. henselae was isolated by screening a genomic DNA cosmid library by Western blotting with sera pooled from patients positive for B. henselae IgG antibodies by indirect immunofluorescence (IFA). The deduced amino acid sequence of the 43.7 kDa encoded protein was found to be 76.3 % identical to the dihydrolipoamide succinyltransferase enzyme (SucB) of Brucella melitensis. SucB has been shown to be an immunogenic protein during infections by Brucella melitensis, Coxiella burnetii and Bartonella vinsonii. The agreement between reactivity with a recombinant SucB fusion protein on immunoblot analysis and the results obtained by IFA was 55 % for IFA-positive sera and 88 % for IFA-negative sera. Cross-reactivity was observed with sera from patients with antibodies against Brucella melitensis, Mycoplasma pneumoniae, Francisella tularensis, Coxiella burnetii and Rickettsia typhi.

The purpose of the present study was to investigate the diversity of the genes encoding aminoglycoside-modifying enzymes and their association with class 1 integrons in three pan-European clones of Acinetobacter baumannii. The study collection included 106 multidrug-resistant strains previously allocated to clone I (n = 56), clone II (n = 36) and clone III (n = 6) and a heterogeneous group of other strains (n = 8), using AFLP fingerprinting and ribotyping. The strains were from hospitals of the Czech Republic (n = 70; collected 1991–2001) and 12 other European countries (n = 36; 1982–1998). Using PCR, at least one of the following aminoglycoside-resistance genes was detected in 101 (95 %) strains: aphA1 (n = 76), aacC1 (n = 68), aadA1 (n = 68), aphA6 (n = 55), aadB (n = 31), aacC2 (n = 7) and aacA4 (n = 3). A combination of two to five different resistance genes was observed in 89 strains (84 %), with a total of 12 different combinations. PCR mapping revealed that aacC1, aadA1 and aacA4 were each associated with a class 1 integron, as was the case with aadB for six strains of clone III. Six different class 1 integron variable regions were detected in 78 strains (74 %), with two predominant regions (2.5 and 3.0 kb) in two sets of 34 strains each. The 3.0 kb region contained five gene cassettes (aacC1, orfX, orfX, orfX′, aadA1) and differed from the 2.5 bp region only by one additional orfX cassette. These two integron regions were confined to clones I and II and were found in strains isolated in seven countries between 1982 and 2001. The clone III strains were homogeneous both in resistance genes and in integron variable regions, whereas clones I and II showed a remarkable intraclonal diversity of these properties, with no clear-cut difference between the two clones. Yet, within the Czech clone I and II strains, the diversity of resistance genes and integron structures was limited as compared to those from other countries. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.

A screw-capped glass tube containing a Leptospira culture accidentally broke and the laboratory worker who was handling the tube sustained a cut on his hand. The wound was flooded with the culture. The culture was that of strain MG 347 belonging to serovar Australis recovered from a patient, and it had undergone 52 passages in Ellinghausen McCullough Johnson Harris medium. The laboratory worker developed a headache 21 days after the accident and became febrile the next day. He was hospitalized for 5 days and was treated initially with doxycycline and later with ciprofloxacin. A blood sample collected on the second day of illness, after starting doxycycline therapy, yielded leptospires and the isolate, HZ 651, was identified as serovar Australis. Monoclonal antibody patterns and randomly amplified polymorphic DNA fingerprinting patterns of the isolate and strain MG 347 were identical, thus indicating that HZ 651 and MG 347 were clonal.