Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.     

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires'' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae''s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.     

Development and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera

The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%).This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture.Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.     

Intracellular Proliferation of Legionella pneumophila in Hartmannella vermiformis in Aquatic Biofilms Grown on Plasticized Polyvinyl Chloride

The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-μm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm²) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% ± 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation.     

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

Aims: To optimize ethidium monoazide (EMA) coupled with real‐time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. Methods and Results: EMA (0·9–45·5 μg ml^?1) and propidium monoazide (PMA, 0·9 and 2·3 μg ml^?1) combined with qPCR (i.e. EMA‐qPCR and PMA‐qPCR, respectively) were applied to unheated and heated (70°C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight‐EM). The effects of nontarget microflora and sample matrix on the performance of EMA‐qPCR were also evaluated. In comparison with BacLight‐EM results, qPCR with EMA at 2·3 μg ml^?1 was determined as the optimal EMA‐qPCR assay, which performed equally well as PMA‐qPCR for unheated Leg. pneumophila but better than PMA‐qPCR for heated Leg. pneumophila (P < 0·05). Moreover, qPCR with EMA at 2·3 μg ml^?1 accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella‐like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0·05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA‐qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. Conclusions: The qPCR with EMA at 2·3 μg ml^?1 may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. Significance and Impact of the Study: The EMA‐qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.     

Necrotrophic Growth of Legionella pneumophila

This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 ± 0.32 log units (n = 5) for real-time PCR and 1.14 ± 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.     

Pulmonary Collectins Protect Macrophages against Pore-forming Activity of Legionella pneumophila and Suppress Its Intracellular Growth

Pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate immunity of the lung. Legionella pneumophila is a bacterial respiratory pathogen that can replicate within macrophages and causes opportunistic infections. L. pneumophila possesses cytolytic activity, resulting from insertion of pores in the macrophage membrane upon contact. We examined whether pulmonary collectins play protective roles against L. pneumophila infection. SP-A and SP-D bound to L. pneumophila and its lipopolysaccharide (LPS) and inhibited the bacterial growth in a Ca²⁺-dependent manner. The addition of LPS in the culture blocked the inhibitory effects on L. pneumophila growth by the collectins, indicating the importance of LPS-collectin interaction. When differentiated THP-1 cells were infected with L. pneumophila in the presence of SP-A and SP-D, the number of permeable cells was significantly decreased, indicating that pulmonary collectins inhibit pore-forming activity of L. pneumophila. The number of live bacteria within the macrophages on days 1–4 after infection was significantly decreased when infection was performed in the presence of pulmonary collectins. The phagocytosis experiments with the pH-sensitive dye-labeled bacteria revealed that pulmonary collectins promoted bacterial localization to an acidic compartment. In addition, SP-A and SP-D significantly increased the number of L. pneumophila co-localized with LAMP-1. These results indicate that pulmonary collectins protect macrophages against contact-dependent cytolytic activity of L. pneumophila and suppress intracellular growth of the phagocytosed bacteria. The promotion of lysosomal fusion with Legionella-containing phagosomes constitutes a likely mechanism of L. pneumophila growth suppression by the collectins.     

Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.     

Necessity and Effect of Combating Legionella pneumophila in Municipal Shower Systems

The objective was to obtain research-based, holistic knowledge about necessity and effect of practiced measures against L. pneumophila in municipal shower systems in Stavanger, Norway. The effects of hot water treatment and membrane-filtering were investigated and compared to no intervention at all. The studies were done under real-world conditions. Additionally, a surveillance pilot study of municipal showers in Stavanger was performed. The validity of high total plate count (TPC) as an indication of L. pneumophila was evaluated. A simplified method, named “dripping method”, for detection and quantification of L. pneumophila was developed. The sensitivity of the dripping method is 5 colony-forming units of L. pneumophila/ml. The transference of L. pneumophila from shower water to aerosols was studied. Interviews and observational studies among the stakeholders were done in order to identify patterns of communication and behavior in a Legionella risk perspective. No substantial effects of the measures against L. pneumophila were demonstrated, except for a distally placed membrane filter. No significant positive correlation between TPC and L. pneumophila concentrations were found. L. pneumophila serogroup 2–14 was demonstrated in 21% of the 29 buildings tested in the surveillance pilot. Relatively few cells of L. pneumophila were transferred from shower water to aerosols. Anxiety appeared as the major driving force in the risk governance of Legionella. In conclusion, the risk of acquiring Legionnaires'' disease from municipal shower systems is evaluated as low and uncertain. By eliminating ineffective approaches, targeted Legionella risk governance can be practiced. Risk management by surveillance is evaluated as appropriate.     

Immunolocalization of Hsp60 in Legionella pneumophila

One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including L. pneumophila, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L. pneumophila grown in vitro, or in vivo inside of HeLa cells. For comparative purposes Escherichia coli, expressing recombinant L. pneumophila Hsp60, was employed. In contrast to E. coli, where Hsp60 was localized exclusively in the cytoplasm, in L. pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked L. pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by L. pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in L. pneumophila and further suggest both the existence of a novel secretion mechanism (not present in E. coli) and a potential role in pathogenesis.     

Identification of Host Cytosolic Sensors and Bacterial Factors Regulating the Type I Interferon Response to Legionella pneumophila

Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires'' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN) in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.     

Legionella pneumophila Persists within Biofilms Formed by Klebsiella pneumoniae,Flavobacterium sp., and Pseudomonas fluorescens under Dynamic Flow Conditions

Legionella pneumophila, the agent of Legionnaires'' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×10⁴ CFU per cm² of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.     

Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires'' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires'' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila.     

Evaluation of the Duopath Legionella Lateral Flow Assay for Identification of Legionella pneumophila and Legionella Species Culture Isolates

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries’ regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.     

The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.     

Presence and Persistence of Legionella spp. in Groundwater

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.     

Isotopologue Profiling of Legionella pneumophila: ROLE OF SERINE AND GLUCOSE AS CARBON SUBSTRATES*

Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires'' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-¹³C₃]serine, [U-¹³C₆]glucose, or [1,2-¹³C₂]glucose. After growth, ¹³C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-¹³C₃]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [¹³C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-¹³C₂]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Δzwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.     

<Emphasis Type="Italic">Legionella</Emphasis> pneumonia due to non-<Emphasis Type="Italic">Legionella pneumophila</Emphasis> serogroup 1: usefulness of the six-point scoring system

Background

Because of a limited number of reports, we aimed to investigate the clinical characteristics of patients with Legionella pneumonia due to non-Legionella pneumophila serogroup 1 and the diagnostic usefulness of the six-point scoring system for such patients compared with patients with pneumonia caused by L. pneumophila serogroup 1.

Methods

We retrospectively analysed patients diagnosed with Legionella pneumonia due to non-L. pneumophila serogroup 1 between March 2001 and June 2016. We examined the clinical characteristics, including symptoms, laboratory findings, radiologic findings, pneumonia severity, initial treatment and prognosis. We also calculated scores using the six-point scoring system in these patients. Furthermore, we compared the clinical characteristics and six-point scores between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients among hospitalized community-acquired pneumonia patients enrolled prospectively between October 2010 and July 2016.

Results

Eleven patients had pneumonia due to non-L. pneumophila serogroup 1; their median age was 66 years and 8 patients (72.7%) were male. The most common pathogen was L. pneumophila serogroup 3 (6/11), followed by L. pneumophila serogroup 9 (3/11), L. pneumophila serogroup 6 (1/11) and L. longbeachae (1/11). Non-specific symptoms, such as fever and cough, were common. Six patients (54.5%) had liver enzyme elevation, but no patient developed hyponatraemia at <130 mEq/L. Nine patients (81.8%) showed lobar pneumonia and 7 patients (63.6%) manifested with consolidation and ground-glass opacity. Patients with mild to moderate severity comprised 10 (90.9%) by CURB-65 and 5 (45.5%) by the Pneumonia Severity Index. Of all patients, 4 were admitted to the intensive care unit and 3 died despite appropriate empiric therapy. The clinical characteristics were not significantly different between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients (n?=?23). At a cut-off value of ≥?2 points, the sensitivity of the six-point scoring system was 54.5% (6/11) for non-L. pneumophila serogroup 1 patients and 95.7% (22/23) for L. pneumophila serogroup 1 patients.

Conclusions

Cases of non-L. pneumophila serogroup 1 pneumonia varied in severity from mild to severe and the clinical characteristics were often non-specific. The six-point scoring system was not useful in predicting such Legionella pneumonia cases.