Novel UL23 and UL30 substitutions in HSV1 and HSV2 viruses related to polymorphism or drug resistance.

Data on herpes simplex virus (HSV) polymorphism as well as acyclovir (ACV) and foscarnet (FOS) resistance mutations are not exhaustive and may hinder accurate diagnosis by next-generation sequencing (NGS). Here, we report novel UL23 and UL30 substitutions for HSV1 and HSV2 identified in immunocompromised patients treated for hematological malignancies during the last 6 years of HSV resistance surveillance at the University Hospital of Lyon. For HSV1, 35 novel UL23 substitutions and 52 novel UL30 substitutions were identified. For HSV2, 2 novel UL23 substitutions and 12 novel UL30 substitutions were identified. These results allow to complete the database of HSV1 and HSV2 substitutions, related either to polymorphism or to ACV and FOS resistance.

A real-time PCR for specific detection of the Legionella pneumophila serogroup 1 ST1 complex.

OBJECTIVE: Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment. METHODS: A total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster. RESULTS: Of seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1-99.9)) and specificity of 100% (122/122) (95% CI (97-100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation. CONCLUSION: Using whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.

Adaptation of Amoeba Plate Test To Recover Legionella Strains from Clinical Samples.

The isolation of Legionella from respiratory samples is the gold standard for diagnosis of Legionnaires' disease (LD) and enables epidemiological studies and outbreak investigations. The purpose of this work was to adapt and to evaluate the performance of an amoebic coculture procedure (the amoeba plate test [APT]) for the recovery of Legionella strains from respiratory samples, in comparison with axenic culture and liquid-based amoebic coculture (LAC). Axenic culture, LAC, and APT were prospectively performed with 133 respiratory samples from patients with LD. The sensitivities and times to results for the three techniques were compared. Using the three techniques, Legionella strains were isolated in 46.6% (n = 62) of the 133 respiratory samples. The sensitivity of axenic culture was 42.9% (n = 57), that of LAC was 30.1% (n = 40), and that of APT was 36.1% (n = 48). Seven samples were positive by axenic culture only; for those samples, there were <10 colonies in total. Five samples, all sputum samples, were positive by an amoebic procedure only (5/5 samples by APT and 2/5 samples by LAC); all had overgrowth by oropharyngeal flora with axenic culture. The combination of axenic culture with APT yielded a maximal isolation rate (i.e., 46.6%). Overall, the APT significantly reduced the median time for Legionella identification to 4 days, compared with 7 days for LAC (P < 0.0001). The results of this study support the substitution of LAC by APT, which could be implemented as a second-line technique for culture-negative samples and samples with microbial overgrowth, especially sputum samples. The findings provide a logical basis for further studies in both clinical and environmental settings.

Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47.

OBJECTIVES: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain. METHODS: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47. RESULTS: The gene LPO_1073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO_1073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO_1073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain. CONCLUSION: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

Molecular epidemiology, phylogeny and evolution of Legionella.

Legionella are opportunistic pathogens that develop in aquatic environments where they multiply in protozoa. When infected aerosols reach the human respiratory tract they may accidentally infect the alveolar macrophages leading to a severe pneumonia called Legionnaires' disease (LD). The ability of Legionella to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Although Legionella is a large genus comprising nearly 60 species that are worldwide distributed, only about half of them have been involved in LD cases. Strikingly, the species Legionella pneumophila alone is responsible for 90% of all LD cases. The present review summarizes the molecular approaches that are used for L. pneumophila genotyping with a major focus on the contribution of whole genome sequencing (WGS) to the investigation of local L. pneumophila outbreaks and global epidemiology studies. We report the newest knowledge regarding the phylogeny and the evolution of Legionella and then focus on virulence evolution of those Legionella species that are known to have the capacity to infect humans. Finally, we discuss the evolutionary forces and adaptation mechanisms acting on the Dot/Icm system itself as well as the role of mobile genetic elements (MGE) encoding T4ASSs and of gene duplications in the evolution of Legionella and its adaptation to different hosts and lifestyles.

Epidemiologic characteristics associated with ST23 clones compared to ST1 and ST47 clones of Legionnaires disease cases in France.

In France, approximately 1200 cases of Legionnaires disease (LD) are reported annually, and isolates are available for approximately 20% of cases identified since 2000. All Legionella pneumophila serogroup 1 (sg1) isolates are characterized by sequence-based typing at the National Reference Centre. LD cases caused by L. pneumophila sg1 reported from 2008 through 2012 were considered for the study. Our study objective was to describe cases according to their sequence type (ST). We also constructed multivariable modified Poisson regression models to estimate the incidence rate ratio (IRR) and to identify characteristics potentially associated with ST23 clones compared to ST1 and ST47 clones. We studied 1192 patients infected by ST1 (n = 109), ST23 (n = 236), ST47 (n = 123) or other STs (n = 724). The geographic distribution of the ST23 cases across the country was significantly different compared to other ST groups. This genotype was significantly associated with the absence of corticosteroid therapy compared to ST1 (IRR = 0.56; p 0.016). Concerning exposure, the ST23 genotype was significantly less associated with hospital-acquired infections compared to ST1 (IRR = 0.32; p 0.001), but it was more associated with infections acquired in hospitals and elderly settings compared with ST47. Finally, the ST23 genotype was less frequently associated with travel than other STs. Despite the large number of cases of ST23 infection, we did not identify any characteristics specific to this ST. However, we identified independent associations between ST1 and nosocomial transmission and steroid therapy. These findings should encourage further exploration, especially in terms of environmental diffusion, strain virulence and host factors.

Evaluation of BMPA, MWY, GVPC and BCYE media for the isolation of Legionella species from respiratory samples.

Culture media performance is a critical factor in the isolation of Legionellae from respiratory samples. We showed that BMPA and MWY media yielded significantly higher isolation rates than GVPC and BCYE media in regard to performance with samples that harbored low Legionella inocula and high contamination levels.

Characterization of a novel composite staphylococcal cassette chromosome mec (SCCmec-SCCcad/ars/cop) in the neonatal sepsis-associated Staphylococcus capitis pulsotype NRCS-A.

Multiresistant Staphylococcus capitis pulsotype NRCS-A has been reported to be a major pathogen causing nosocomial bacteremia in preterm infants. We report that the NRCS-A strain CR01 harbors a novel 60.9-kb composite staphylococcal cassette chromosome mec (SCCmec) element, composed of an SCCmec with strong homologies to Staphylococcus aureus ST398 SCCmec and of an SCCcad/ars/cop harboring resistance genes for cadmium, arsenic, and copper. Whole-genome-based comparisons of published S. capitis strains suggest that strain CR01 acquired the two elements independently.

Contribution of amoebic coculture to recovery of Legionella isolates from respiratory samples: prospective analysis over a period of 32 months.

We evaluated the contribution of amoebic coculture to the recovery of Legionella spp. from 379 respiratory samples. The sensitivity of axenic culture was 42.1%. The combination of axenic culture with amoebic coculture increased the Legionella isolation rate to 47.1%. Amoebic coculture was particularly efficient in isolating Legionella spp. from respiratory samples contaminated with oropharyngeal flora.

Impact of the virulence-associated MAb3/1 epitope on the physicochemical surface properties of Legionella pneumophila sg1: An issue to explain infection potential?

The relationship between the presence/absence of the virulence-associated MAb3/1 epitope of sixteen Legionella pneumophila serogroup 1 strains and their respective surface physicochemical properties is evidenced from electrokinetic measurements (microelectrophoresis) performed as a function of KNO(3) electrolyte concentration (range 1-100mM, pH∼6.5). Among the bacteria selected, nine original strains constitute the Dresden reference panel and differ according to the presence/absence of the virulence-associated monoclonal antibody MAb3/1 of the O-specific chain of the lipopolysaccharides (LPS). Five isogenic Lens strains, also investigated in the current study, present the epitope MAb3/1 of their LPS and were involved to some extent in the outbreak that stroke the Nord Pas-de-Calais region (France) in 2004. All bacteria exhibit the typical electrokinetic features of soft (permeable) particles. On the basis of Ohshima's model, analysis of the electrophoretic mobility data allows evaluating the intraparticular flow penetration length 1/λ(0) and the (negative) volume charge density ρ(0) that both reflect the structure and chemical composition of the soft bacterial component. Our results show that the virulent MAb3/1 positive strains are characterized on average by 1/λ(0) and ǀρ(0)ǀ values that are about 1.5 times larger and 5 times lower, respectively, than those derived for lesser virulent (MAb3/1 negative) strains. In other words, on average the soft surface layer of MAb3/1 positive strains is significantly less charged and more permeable than those of MAb3/1 negative strains. The intimate correlation between virulence-associated MAb3/1 epitope and charge density carried by the bacterial envelop was further confirmed by lower 1/λ(0) and greater ǀρ(0)ǀ values for lag-1 mutant CS332 strain, lacking the MAb3/1 epitope, compared to the parental strain AM511. A closer inspection of the dispersion in 1/λ(0) and ǀρ(0)ǀ data over the ensemble of analysed bacteria together with the reported number of Legionnaires' disease cases they are responsible for, points out the charge density ǀρ(0)ǀ as the parameter that is most suitable for discriminating highly virulent (MAb3/1 positive) from less virulent (MAb3/1 negative) strains. Although short-range interaction determines infection process, our results suggest that the infection potential of Legionella pneumophila serogroup 1 may be also controlled significantly by non-specific long-range electrostatic repulsion the bacteria undergo when approaching negatively charged host cells to be infected.

Development and evaluation of Chlamylege, a new commercial test allowing simultaneous detection and identification of Legionella, Chlamydophila pneumoniae, and Mycoplasma pneumoniae in clinical respiratory specimens by multiplex PCR.

This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 x 10(-3) IFU, 5 x 10(-2) color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses.

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.