Monoclonal Antibody Subgrouping Research Articles

Population structure of Environmental and Clinical Legionella pneumophila isolates in Catalonia

Legionella is the causative agent of Legionnaires’ disease (LD). In Spain, Catalonia is the region with the highest incidence of LD cases. The characterisation of clinical and environmental isolates using molecular epidemiology techniques provides epidemiological data for a specific geographic region and makes it possible to carry out phylogenetic and population-based analyses. The aim of this study was to describe and compare environmental and clinical isolates of Legionella pneumophila in Catalonia using sequence-based typing and monoclonal antibody subgrouping. A total of 528 isolates were characterised. For data analysis, the isolates were filtered to reduce redundancies, and 266 isolates (109 clinical and 157 environmental) were finally included. Thirty-two per cent of the clinical isolates were ST23, ST37 and ST1 while 40% of the environmental isolates were ST284 and ST1. Although the index of diversity was higher in clinical than in environmental ST isolates, we observed that clinical STs were similar to those recorded in other regions but that environmental STs were more confined to particular study areas. This observation supports the idea that only certain STs trigger cases or outbreaks in humans. Therefore, comparison of the genomes of clinical and environmental isolates could provide important information about the traits that favour infection or environmental persistence.

Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene

Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires‘ disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.

Molecular typing of Legionella pneumophila serogroup 1 clinical strains isolated in Italy

Molecular typing methods for discriminating different bacterial isolates are essential epidemiological tools in prevention and control of Legionella infections and outbreaks. A selection of 56 out of 184 Legionella pneumophila serogroup 1 (Lp1) clinical isolates, collected from different Italian regions between 1987 and 2012, and stored at the National Reference Laboratory for Legionella, were typed by monoclonal antibody (MAb) subgrouping, amplified fragment length polymorphism (AFLP) and sequence based typing (SBT). These strains were isolated from 39 community (69.6%), 14 nosocomial (25%) and 3 travel associated (5.4%) Legionnaires’disease cases. MAb typing results showed a prevalence of MAb 3/1 positive isolates (75%) with the Philadelphia subgroup representing 35.7%, followed by Knoxville (23.2%), Benidorm (12.5%), Allentown/France (1.8%), Allentown/France-Philadelphia (1.8%). The remaining 25% were MAb 3/1 negative, namely 11 Olda (19.6%), 2 Oxford (3.6%) and 1 Bellingham (1.8%) subgroups.AFLP analysis detected 20 different genomic profiles. SBT analysis revealed 32 different sequence types (STs) with high diversity of STs (IODSTs=0.952) 12 of which were never described before. ST1 and ST23 were most frequently isolated as observed worldwide. A helpful analysis of data from SBT, MAb subgrouping and AFLP is provided, as well as a comparison to the Lp1 types investigated from other countries. This study describes the first Italian Lp1 strains database, providing molecular epidemiology data useful for future epidemiological investigations, especially of travel associated Legionnaires’ diseases (TALD) cases, Italy being the country associated with the highest number of clusters.

Distribution of Monoclonal Antibody Subgroups and Sequence-Based Types among Legionella pneumophila Serogroup 1 Isolates Derived from Cooling Tower Water, Bathwater, and Soil in Japan

Legionella pneumophila serogroup (SG) 1 is the most frequent cause of legionellosis. This study analyzed environmental isolates of L. pneumophila SG 1 in Japan using monoclonal antibody (MAb) typing and sequence-based typing (SBT). Samples were analyzed from bathwater (BW; n = 50), cooling tower water (CT; n = 50), and soil (SO; n = 35). The distribution of MAb types varied by source, with the most prevalent types being Bellingham (42%), Oxford (72%), and OLDA (51%) in BW, CT, and SO, respectively. The ratios of MAb 3/1 positive isolates were 26, 2, and 14% from BW, CT, and SO, respectively. The environmental isolates from BW, CT, and SO were divided into 34 sequence types (STs; index of discrimination [IOD] = 0.973), 8 STs (IOD = 0.448), and 11 STs (IOD = 0.879), respectively. Genetic variation among CT isolates was smaller than seen in BW and SO. ST1 accounted for 74% of the CT isolates. The only common STs between (i) BW and CT, (ii) BW and SO, and (iii) CT and SO were ST1, ST129, and ST48, respectively, suggesting that each environment constitutes an independent habitat.

Insertion Sequences as Highly Resolutive Genomic Markers for Sequence Type 1 Legionella pneumophila Paris

The causative agent of legionellosis, Legionella pneumophila, colonizes all natural and human-made water networks, thus constituting the source of contaminated aerosols responsible for airborne human infections. Efficient control of infections, especially during epidemics, necessitates the fastest and most resolutive identification possible of the bacterial source for subsequent disinfection of reservoirs. We thus compared recognized typing approaches for Legionella with a method based on characterization of insertion sequence (IS) content. A total of 86 clinical or environmental isolates of L. pneumophila, including 84 Paris isolates, sampled from 25 clinical investigations in France between 2001 and 2007, were obtained from the Legionella National Reference Center. All strains were typed by monoclonal antibody subgrouping, sequence-based typing, pulsed-field gel electrophoresis, and restriction fragment length polymorphism based on the presence or absence of IS elements. We identified six different types of IS elements in L. pneumophila Paris and used them as genomic markers in hybridization experiments. One IS type, ISLpn11, revealed a high discriminatory power. Simpson's index of discrimination, calculated from the distribution of IS elements, was higher than that obtained with the other typing methods used for L. pneumophila Paris. Moreover, specific ISLpn11 copies were found only in strains isolated from particular cities. In more than half of the cases, each clinical isolate had an ISLpn11 profile that was recovered in at least one environmental isolate from the same geographical location, suggesting that our method could identify the infection source. Phylogenetic analysis suggests a clonal expansion for the L. pneumophila Paris strain.

Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types

We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980-2008. Most (80.2%) belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95% CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9% of all isolates). Moreover, 79.7% of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulence-associated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n=6), ST120 (n=5) and ST138 (n=5). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies.

Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila

A total of 57 isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody subgrouping, seven-gene locus sequence-based typing (SBT) scheme and a newly developed variable element typing (VET) system based on the presence or absence of ten variable genetic elements. These elements were detected while screening a genomic library of strain Corby, as well as being taken from published data for PAI-1 (pathogenicity island) from strain Philadelphia. Specific primers were designed and used in gel-based polymerase chain reaction (PCR) assays. PCR amplification of the mip gene served as a control. The end-point was the presence/absence of a PCR product on an ethidium bromide-strained gel. In the present study, the index of discrimination was somewhat lower than that of the SBT (0.87 versus 0.97). Nevertheless, the results obtained showed as a 'proof of principle' that this simple and quick typing assay might be useful for the epidemiological characterisation of L. pneumophila strains.

Legionella pneumophila monoclonal antibody subgroups and DNA sequence types isolated in Canada between 1981 and 2009: Laboratory Component of National Surveillance

Legionella pneumophila (Lp) is a significant cause of nosocomial, community-acquired, and travel-associated pneumonia in industrialized regions. Legionellosis has been a nationally notifiable disease in Canada since 1986, with an average of 75 cases reported annually; however, only the most severe, and often fatal, cases are reported or investigated. Here, epidemiological relationships, types, and distribution of Lp referrals to the Canadian national reference center were studied. Lp strains from different years, sources, and geographic locations were subtyped using a sequence-based typing (SBT) scheme and by the 'Joly' and/or 'Dresden' monoclonal antibody panels. Included were 128 epidemiologically unrelated clinical and 86 unrelated environmental strains. Sixty-four (index of diversity [IOD] = 0.964) and 45 (IOD = 0.888) sequence types (STs) were observed among clinical and environmental sources, respectively. Serogroup (sg) 1 was represented by 60.2% (77/128) and 52.3% (45/86) of clinical and environmental strains, respectively, and 63.6% (49/77) and 15.6% (7/45) of those were mAb2-positive, respectively. Serogroup 1, ST1 accounted for 14.1% (18/128) and 30.2% (26/86) of unrelated clinical and environmental isolates, respectively. This database will serve as a basis for Canadian epidemiological surveillance efforts and is linked to global surveillance initiatives curated by the European Working Group for Legionella Infections (EWGLI) network.

Distribution of Legionella pneumophila serogroups, monoclonal antibody subgroups and DNA sequence types in recent clinical and environmental isolates from England and Wales (2000–2008)

Clinical isolates of Legionella pneumophila, obtained from 167 patients, who acquired their illness in the community in England and Wales between January 2000 and March 2008, were compared with 276 environmental isolates of L. pneumophila obtained over the same period as part of the routine sampling of 'managed' water systems. The 443 isolates were typed by monoclonal antibody (mAb) subgrouping and the internationally standardised, seven-gene loci, sequence-based typing (SBT) scheme of the European Working Group for Legionella Infections (EWGLI). Of the clinical isolates, 97.6% were L. pneumophila serogroup (sgp) 1, compared with only 55.8% of environmental isolates (P = 0.0002); 91.6% were subgrouped as mAb3/1+ve, compared with only 8.3% of environmental isolates (P < 0.0001). The isolates were very diverse, with SBT identifying 111 sequence types (STs) (index of diversity [IOD] 0.954). Among the clinical isolates, 42 ST were seen, with one (ST47) accounting for 25.7% and three (ST47, ST37 and ST62) accounting for 46.1% of all isolates. Eighty-two STs were identified among the environmental isolates, with two (ST1 and ST79) accounting for 34.1% of these. Comparison of the STs seen among clinical and environmental isolates showed that there was very little overlap between the two populations (P < 0.0001), with common clinical strains found in the environment very infrequently: 0.4, 0.7 and 0% (ST47, ST37 and ST62, respectively), and common environmental strains rarely causing disease: 4.8 and 1.2% (ST1 and ST79, respectively). Combining phenotypic and genotypic data identified 144 phenons (IOD 0.970); 52 among clinical isolates and 101 among environmental isolates. The most abundant clinical strain, mAb 'Allentown' ST47, accounted for 22.8% of cases, but was only found once in the environment. Conversely, mAb 'Oxford/OLDA' ST1 was the most common environmental strain (17.0%), but only caused two infections. A review of the published data shows that mAb 'Allentown' ST47 is also an important cause of infection in France and possibly in the Netherlands. However, it was not found in a large study of German clinical isolates. This study confirms previous work showing that just a few strains of L. pneumophila cause the majority of community-acquired Legionella infection in England and Wales, and that these clinically significant strains are only rarely found in managed water systems. These data suggest that knowing which particular strain is present in an environment might be at least as important as knowing the quantity in which legionellae are present.

Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates ofLegionella pneumophila

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Sequence-based typing of Legionella pneumophila serogroup 1 offers the potential for true portability in legionellosis outbreak investigation.

Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.

Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella Urin Antigen EIA

The new BinaxNOW Immunochromatographic (ICT) Assay for the detection of Legionella pneumophila antigens was used to test 535 urine specimens from patients with and without Legionnaires' disease. The specificity, calculated by testing 112 samples from patients with pneumonia of aetiologies other than Legionella infection, and 167 urine specimens from urinary tract infections, was found to be 97.1% if the manufacturer's guidelines were followed. However, it was determined that the 'false positive' results characterised by very weak bands could be discounted by re-examination of the results at 60 min, yielding a specificity of 100%. With this minor modification of the procedure applied to examination of urine samples from 117 patients with legionellosis confirmed by isolation of L. pneumophila and 70 patients who had seroconverted to L. pneumophila serogroup 1, sensitivity was calculated to be 79.7%. In comparison, the sensitivities of the Binax Urinary Antigen Enzyme Immunoassay (EIA) and Biotest Urin Antigen EIA were estimated to be 79.1 and 83.4%, respectively. Eleven cases (5.9%) were positive by BinaxNOW assay but negative by Binax or Biotest EIA, or both. The sensitivities of all assays increased to c. 94% if only diagnosis of cases confirmed by isolation of serogroup 1 L. pneumophila was considered, although the sensitivity for infections caused by L. pneumophila serogroup 1 monoclonal antibody (MAb) subgroup Bellingham was significantly lower than for other MAb subgroups. The Biotest EIA recognised 10 (45%) of the 22 cases not caused by L. pneumophila serogroup 1, whereas the two Binax kits detected only three each. The ICT assay BinaxNOW can be recommended as a rapid specific test for the diagnosis of Legionnaires' diseases caused by L. pneumophila serogroup 1, although very weak bands should be interpreted cautiously.

Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of São Miguel, Azores

Nineteen aquatic environment sites from three hydrothermal areas on continental Portugal and one area on the island of São Miguel, Azores, were examined for the recovery of Legionella spp. Physicochemical and bacteriological parameters were also determined for each site. Water temperatures varied between 22 and 67.5 degrees C, although the majority had temperatures above 40 degrees C; the pH varied between 5.5 and 9.2. The number of Legionella spp. recovered varied between 5.0 x 10(2) and 2.3 x 10(6) CFU/liter. A total of 288 isolates from 14 sites were identified by indirect immunofluorescence assay. The majority of the isolates belonged to Legionella pneumophila (74.3%), of which most belong to serogroup 1, but the relative proportion of L. pneumophila serogroups varied considerably. L. pneumophila serogroup 1 constituted 96.2% of the isolates in area 2 from central Portugal, but no isolates of this serogroup were recovered from São Miguel, where serogroup 6 strains were the predominant isolates. Ninety-six percent of the L. pneumophila serogroup 1 isolates belonged to monoclonal antibody subgroups OLDA and Bellingham. Other species identified were L. bozemanii serogroup 2, L. dumoffii, L. micdadei, L. moravica, L. oakridgensis, L. sainticrucis, and L. sainthelensi. Two undescribed species, which react by indirect immunofluorescence assay to antisera to "L. londoniensis" and "L. nautarum" and a group of isolates with strong cross-reaction to L. cincinnatiensis/L. sainticrucis/L. longbeachae by indirect immunofluorescence assay were also recovered. The latter were the only isolates recovered from area 3, in east central Portugal, over a period of 1 year.

Phenotypic variation amongst genotypically homogeneousLegionella pneumophilaserogroup 1 isolates: implications for the investigation of outbreaks of Legionnaires' disease

One hundred and seventy-nine isolates of Legionella pneumophila serogroup 1, obtained from a site associated with an outbreak of Legionnaires' disease, were examined by monoclonal antibody subgrouping, restriction fragment length polymorphism typing, restriction endonuclease analysis and plasmid content. Nine distinct phenotypes were detected but at the genotypic level all strains were closely related. The data presented indicate that phenotypic variation of a single parent strain can occur within an environmental site. The implications of these findings are discussed in relation to the investigation of outbreaks of Legionnaires' disease.

Legionella pneumophila in a hospital water system following a nosocomial outbreak: prevalence, monoclonal antibody subgrouping and effect of control measures

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Legionella pneumophila in a hospital water system following a nosocomial outbreak: prevalence, monoclonal antibody subgrouping and effect of control measures.

Swabs and water samples from a hospital water system were cultured for legionellae over an extended period. Legionella pneumophila serogroup 1, including outbreak associated strains, were isolated in small numbers from approximately 5% of these samples despite implementation of the current DHSS/Welsh Office regulations. No cases of nosocomial legionnaires' disease were proven during the study. Physical cleaning and chemical sterilization of taps, and replacement of washers with 'approved' brands did not eradicate the organisms. Eradication of legionellae in hospital water supplies appears to be unnecessary in preventing nosocomial legionnaires' disease provided the current DHSS/Welsh Office recommendations are implemented.