Immunohistochemical Labelling for Cyclo-oxygenase-2: Does the Positive Control Guarantee Standardized Results?

Since the identification of cyclo-oxygenase-2 as a potentially important therapeutic target in veterinary oncology, numerous studies on its expression have been conducted. Unfortunately, results have been heterogeneous and conclusions are difficult to draw. We tested the ability of a defined positive control to guarantee reproducibility of results among different laboratories. Valid positive controls were defined by positivity of the renal macula densa without background labelling. Fifteen colorectal tumours and 15 oral squamous cell carcinomas were labelled immunohistochemically by six European laboratories. Slides were evaluated in blinded fashion for percentage of positive cells and labelling intensity by three pathologists, and results were analyzed statistically for reproducibility and inter-reader variability. Macula densa positivity was an insufficiently sensitive control to guarantee reproducible results for percentage of positive cells and labelling intensity. Inter-reader variability was proven statistically, making the case for image analysis or other automated quantitative evaluation techniques.

Evaluation of an immunomagnetic separation assay in combination with cultivation to improve Legionella pneumophila serogroup 1 recovery from environmental samples.

AIMS: Legionella isolation from environmental samples is often difficult because of the presence of heterotrophic-associated bacteria that frequently overgrow when using standard culture (ISO 11731, 1998; NF T90-431, 2003) methods. To improve Legionella pneumophila recovery from complex water samples (water from cooling towers, biofilms), we evaluated an immunomagnetic separation (IMS) assay using a monoclonal antibody raised against the lipopolysaccharide of Leg. pneumophila sg1 in combination with culture. METHODS AND RESULTS: This study was conducted on 51 environmental specimens. The comparison between IMS-culture and standard culture (ISO 11731, 1998; NF T90-431, 2003) methods was made using ISO 17994, 2004 criteria for establishing equivalence between microbiological methods based on the upper and lower (XH and XL) values of the relative difference (95% confidence limit) and D as maximum acceptable deviation (value of the confidence limit). CONCLUSIONS: We found that the average performance of IMS culture was higher than the reference method.

Investigation of opportunistic pathogens in municipal drinking water under different supply and treatment regimes.

Changing regulations to lower disinfectant byproducts in drinking water is forcing utilities to switch disinfection from chlorine to monochloramine. It is generally unknown whether this will impact positively or negatively on the microbiological quality of drinking water. A utility in Florida, using water with relatively high organic carbon levels from deep wells in several wellfields, made the decision to change its disinfection regime from chlorine to chloramine in order to meet the new regulations. To assess the impacts of such a change on the microbiology of its water supplies, it undertook a number of studies before and after the change. In particular, the presence of the opportunistic pathogens Legionella and Mycobacterium, and also the composition of drinking-water biofilms, were examined. A preliminary synthesis and summary of these results are presented here. Legionella species were widely distributed in source waters and in the distribution system when chlorine was the disinfectant. In some samples they seemed to be among the dominant biofilm bacteria. Following the change to monochloramine, legionellae were not detected in the distribution system during several months of survey; however, they remained detectable at point of use, although with less species diversity. A variety of mycobacteria (21 types) were widely distributed in the distribution system when chlorine was the disinfectant, but these seemed to increase in dominance after chloramination was instituted. At point of use, only four species of mycobacteria were detected. Other changes occurring with chloramination included (a) an altered biofilm composition, (b) increased numbers of total coliforms and heterotrophs and (c) nitrification of water storage tanks. The results suggested that consideration should be given to the microbiological effects of changing disinfection regimes in drinking-water and distribution system biofilms.

Occurrence of Legionella in groundwater: an ecological study.

The natural habitat of Legionella is the water environment. Little is known about their presence in groundwater in spite of the fact that many millions around the globe regularly rely on groundwaters. This pilot study was aimed at evaluating the occurrence of Legionella in groundwater samples (water and biofilms) collected from various sites. Water and biofilm samples from selected groundwater sources were examined for Legionella using culture media (selective and non-selective) and a semi-nested PCR assay. Innovative approaches such as immunomagnetic separation (IMS) in combination with cultivation and flow cytometry were also evaluated. The findings available thus far show that (a) Legionella could be readily recovered from groundwater samples by cultivation even though their numbers showed considerable variations, (b) surprisingly, the PCR methodology was not yet as sensitive as cultivation and (c) flow cytometry was not directly applicable on natural samples because of debris and the high number of heterotrophic associated microflora from which some members were likely to cross-react with the monoclonal antibody used for separation procedures (IMS).

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Isolation of Legionella oakridgensis from two patients with pleural effusion living in the same geographical area.

Two cases of Legionnaire's disease caused by Legionella oakridgensis were diagnosed at the university hospital in Nantes, France. The two patients' isolates were identified by means of phenotyping and genotyping methods. Epidemiological investigations concluded that the first case was hospital acquired while the second case was considered community acquired.

Legionella taurinensis sp. nov., a new species antigenically similar to Legionella spiritensis.

A group of 42 Legionella-like organisms reacting specifically with Legionella spiritensis serogroup 1 antisera were collected throughout Europe by the Centre National de Référence (French National Reference Centre) for Legionella. This group of isolates differed somewhat from L. spiritensis in terms of biochemical reactions, ubiquinone content and protein profile. The latter two analyses revealed that one of these L. spiritensis-like isolates, Turin I no. 1T, was highly related, but not identical to any of the red autofluorescent species of Legionella. In fact, this strain was the first of these particular isolates recognized to emit a red autofluorescence when exposed to UV light. Profile analysis of randomly amplified polymorphic DNA established that the red autofluorescent L. spiritensis-like isolates constituted a homogeneous group distinct from Legionella rubrilucens and Legionella erythra. DNA-DNA hybridization studies involving the use of S1 nuclease confirmed that the indicated group of isolates are a new species of Legionella, for which the name Legionella taurinensis is proposed with strain Turin I no. 1T (deposited as ATCC 700508T) as the type strain.

Identification of Legionella species by random amplified polymorphic DNA profiles.

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5'-CGGCGGCGGCGG-3') and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization.

Species identification of Legionella via intergenic 16S-23S ribosomal spacer PCR analysis.

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests. Amplification polymorphism of the intergenic 16S-23S spacer regions (ISR) has been previously developed for identification of species within the Legionellaceae [Hookey, J.V., Birtles, R.J. & Saunders, N.A. (1995). J Clin Microbiol 33, 2377-2381], but it did not provide enough resolution to distinguish all members of the bluish-white autofluorescent species and the red autofluorescent group of the Legionellaceae. By choosing new primers that target regions 4 (positions 1521-1541 of Escherichia coli 16S rRNA gene) and 6 (positions 114-132 of E.coli 23S rRNA gene) within the rDNA operon close to the 16S-23S intergenic spacer, 34 profiles were determined among the 79 type and reference strains representing 42 species that were tested. Analysis of the RFLP generated after Hinfl restriction digestion of the PCR products further improved the method, allowing complete discrimination among the species and subspecies of Legionella tested. Twenty-three well-identified strains from unrelated origins belonging to seven species gave amplification patterns identical to that of their type strain. The technique was also tested on 80 field isolates that could not be unequivocally assigned to groups by phenotypic methods. Seventy-two per cent (58/80) of these isolates had a profile identical to that of a type strain, while 27% (22/80) may correspond to new taxa since their ISR-PCR profiles did not match any of the known profiles.

Comparative analysis of infrequent-restriction-site PCR and pulsed-field gel electrophoresis for epidemiological typing of Legionella pneumophila serogroup 1 strains.

Two methods were compared for the analysis of 48 unrelated and epidemiologically related Legionella pneumophila serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for XbaI and PstI restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with SfiI. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of L. pneumophila from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, > or = 0.996) tool for typing of Legionella. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.

[Legionnaires' disease in travellers]. / Légionelloses du voyageur.

The outbreak of pneumonia involving delegates to the 1976 American Legion convention at a Philadelphia hotel was the first example of travel-associated legionnaires' disease. Travel is now well known as a common risk factor for legionnaires' disease. This travel-associated disease is a preoccupation among European countries because of morbidity among citizens of the European Union. The definition of the case of legionellosis is a patient who presents an acute lower respiratory tract infection with focal signs of pneumonia and/or radiological features, and microbiological evidence of Legionella infection. A case is considered to be travel associated if the patient has spent one or more nights away from home during the ten days before becoming ill. An European Surveillance Scheme for Travel-Associated Legionnaires' Disease was established in 1987 to identify clusters and outbreaks of cases of the disease. This group centralizes the case reports of twenty-nine collaborating centres in twenty-five countries. Outbreaks of legionnaires' disease were described in hotels, camps or cruise ships. In 1996, the number of travel-associated cases of legionnaires' disease represented 16% of the total number cases. The increase of the number of reported cases may reflect improved surveillance and increased ascertainment. In Europe in 1996, the diagnosis of legionellosis was confirmed by detection of Legionella pneumophila sero-group 1 antigen in urine (36%), seroconversion (fourfold rise in antibody titre, 33%) and culture of the organism (16%). Fifteen per cent of legionellosis was diagnosed by the identification of a single high antibody titre. In France a coordination between Public Health Institutions (Réseau National de Santé Public and DDASS), clinicians, laboratories and National Reference Center was established to improve prevention and control of legionnaires' disease outbreaks. Legislation obliges to report each case. When more two cases in the same area are notified an epidemiological investigation must be done. The knowing of the source of the contamination and its eradication allows to prevent new cases and outbreaks. Outbreaks of Legionnaires' disease are even now mediatic and this fact leads to maintain attention for the quality of diagnosis and epidemiology investigation due to touristic and economic consequences for the implicated countries.

The first clinical isolate of Legionella parisiensis, from a liver transplant patient with pneumonia.

A bluish white autofluorescent strain of Legionella was isolated from the tracheal aspirate of a female liver transplant patient who developed hospital-acquired pneumonia. This strain had biochemical characteristics compatible with those of L. cherrii, L. anisa, and L. parisiensis and could not be differentiated from L. bozemanii and L. parisiensis by the direct fluorescent-antibody assay. Phylogenetic analysis of partial 16S rRNA gene sequences of this strain (ATCC 700174) revealed the closest homology to the species L. parisiensis (99.5%). An L. parisiensis species-specific profile was also identified by a random amplified polymorphic DNA technique. This is the first report of L. parisiensis isolation from humans.

Distribution of mip-related sequences in 39 species (48 serogroups) of Legionellaceae.

The macrophage infectivity potentiator gene (mip) from Legionella pneumophila is a major virulence factor of the species. Thus, mip-detection by amplification has been proposed to assess the presence of L. pneumophila in clinical and environmental samples. The distribution of mip-related sequences within the Legionellaceae was studied by DNA amplification using mip-specific primers followed by Southern blot hybridization with an internal probe. Thirty-nine species (48 serogroups) of Legionellaceae were screened in this attempt. Using this approach, sequences related to mip were observed in 89% of the tested species including the most recently described L. fairfieldensis, L. lansingensis and L. shakespearei. In several cases, cloning and sequencing of the amplified products confirmed the high levels of similarity between the sequence found in non-pneumophila species with that of the L. pneumophila mip gene. This confirms previous reports that mip related genes are widespread among Legionellaceae and therefore specific detection of the species L. pneumophila cannot be based on mip-targeted amplification.