Metodi diagnostici e di laboratorio (2020 - 2017)
[ultimo aggiornamento 25/11/2020]
Evaluation
of Legiolert for Quantification of Legionella
pneumophila from Bath Water Samples
Inoue H,
Baba M, Tayama S.
Tsukuba Research Laboratories, Aquas Corporation, Japan. h_inoue0417@aquas.co.jp
Biocontrol Sci 2020;25(3):179-182.
Abstract:
Testing for Legionella
spp. in public bath water samples is regulated in Japan. In this study, we used
a total of 132 public bath water samples to compare the performance of Legiolert®
and the conventional plate culture method for the enumeration of Legionella
pneumophila. When Legiolert and plate culturing were performed at the same
detection limit, L. pneumophila was
detected in 26.5% of 132 samples by Legiolert, while 12.9% contained Legionella spp. (11.4% contained L. pneumophila) based on the plate culture method. Moreover, results
of 83.3% of the total samples were consistent between the two methods, meaning
that they were both positive or both negative. In this study, we demonstrated
that Legiolert is a simpler and more effective method of monitoring for L.
pneumophila in bath water samples.
Droplet
digital PCR for the detection and monitoring of Legionella pneumophila
Falzone L, Gattuso G,
Lombardo C, Lupo G, Grillo CM, Spandidos DA, Libra M, Salmeri M.
Department of Biomedical and Biotechnological Sciences, Section of
General Pathology, University of Catania. m.libra@unict.it
Int J Mol Med 2020 Nov;46(5):1777-1782.
Abstract:
Legionella
pneumophila (L. pneumophila) is a harmful pathogen often found in water systems.
In hospitals, the absence of L.
pneumophila in water systems is mandatory by law, therefore, frequent and
effective monitoring of water is of fundamental importance. Molecular methods
based on reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR)
have been proposed for the detection of L.
pneumophila, however, the sensitivity and accuracy of these methods have not
been validated yet. Therefore, it is important to evaluate other strategies able
to overcome the limits of culture‑based and RT‑qPCR methods. On
these bases, we compared the sensitivity and accuracy of droplet digital PCR (ddPCR)
and RT‑qPCR in water samples with known concentrations of L. pneumophila and in an in vitro model of water heat treatments.
ddPCR showed a higher sensitivity rate and accuracy compared to RT‑qPCR in
detecting low bacterial load. In addition, ddPCR is not affected by the presence
of fragmented DNA and showed higher accuracy than RT‑qPCR in monitoring
the efficacy of heat shock treatments. In conclusion, ddPCR represents an
innovative strategy to effectively detect L. pneumophila in water samples. Thanks to its high robustness,
ddPCR could be applied also for the detection of L. pneumophila in patients with suspected legionellosis.
Evaluation
of GVPC and BCYE Media for Legionella Detection and Enumeration
in Water Samples by ISO 11731: Does Plating on BCYE Medium Really Improve Yield?
Scaturro M, Poznanski E, Mupo
M, Blasior P, Seeber M, Prast AM, Romanin E, Girolamo A, Rota MC, Bella A, Ricci
ML, Stenico A.
Department of Infectious Diseases, Istituto Superiore di Sanità, Rome,
Italy. marialuisa.ricci@iss.it
Pathogens 2020 Sep 16;9(9):757.
Abstract:
Legionella spp are the causative agents of Legionnaires' diseases, which is a
pneumonia of important public health concern. Ubiquitous freshwater and soil
inhabitants can reach man-made water systems and cause illness. Legionella enumeration
and quantification in water systems is crucial for risk assessment and culture
examination is the gold standard method. In this study, Legionella recovery
from potable water samples, at presumably a low concentration of interfering
microorganisms, was compared by plating on buffered charcoal yeast extract (BCYE)
and glycine, vancomycin, polymyxin B, cycloheximide (GVPC) Legionella agar
media, according to the International Standard Organization (ISO) 11731: 2017.
Overall, 556 potable water samples were analyzed and 151 (27.1%) were positive
for Legionella. Legionella grew on both BCYE and
GVPC agar plates in 85/151 (56.3%) water samples, in 65/151 (43%) on only GVPC
agar plates, and in 1/151 (0.7%) on only BCYE agar plates. In addition, GVPC
medium identified Legionella species other than pneumophila in
six more samples as compared with the culture on BCYE. Although the medians of
colony forming units per liter (CFU/L) detected on the BCYE and GVPC agar plates
were 2500 and 1350, respectively (p-value < 0.0001), the difference
did not exceed one logarithm, and therefore is not relevant for Legionella risk
assessment. These results make questionable the need to utilize BCYE agar plates
to analyze potable water samples.
Performance of the ImmuView and BinaxNOW assays for
the detection of urine and cerebrospinal fluid Streptococcus pneumoniae and Legionella
pneumophila serogroup 1 antigen in patients with Legionnaires' disease or
pneumococcal pneumonia and meningitis
Edelstein PH, Jørgensen CS, Wolf LA.
Department of Pathology and Laboratory Medicine,
Perelman School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania, USA. paul.edelstein@pennmedicine.upenn.edu
PLoS One 2020 Aug 31;15(8):e0238479.
Abstract: The performances of the ImmuView Streptococcus
pneumoniae (Sp) and Legionella pneumophila (Lp) urinary antigen test were
compared to that of the BinaxNOW Sp and Lp assays, using frozen urine from 166
patients with Legionnaires' disease (LD) and 59 patients with pneumococcal
pneumonia. Thirty Sp-positive or contrived cerebrospinal fluids (CSF) were also
tested. Test specimens were collected and tested at different sites, with each
site testing unique specimens by technologists blinded to expected results. No
significant differences in test concordances were detected for the ImmuView and
BinaxNOW assays for the Sp or Lp targets for urine from patients with
pneumococcal pneumonia or LD when performance from both sites were combined. At
one of two test sites the ImmuView Lp assay was more sensitive than the BinaxNOW
assay, with no correlation between test performance and Lp serogroup 1
monoclonal type. Urines from six of seven patients with LD caused by Legionella
spp. bacteria other than Lp serogroup 1 were negative in both assays. Both tests
had equivalent performance for Sp-positive CSF. The clinical sensitivities for
pneumococcal pneumonia were 88.1 and 94.4% for the ImmuView and Binax assays,
and 87.6 and 84.2% for the Lp assays, respectively. Test specificities for
pneumococcal pneumonia were 96.2 and 97.0% for the ImmuView and Binax assays,
and 99.6 and 99.1% for the Lp assays. Both assays were highly specific for Sp in
pediatric urines from children with nasopharyngeal colonization by the bacterium.
ImmuView and BinaxNOW assay performance was equivalent in these studies.
Rapid
Detection of Legionella pneumophila in Drinking Water, Based on
Filter Immunoassay and Chronoamperometric Measurement
Ezenarro
JJ, Párraga-Niño N, Sabrià M, Del Campo FJ, Muñoz-Pascual
FX, Mas J, Uria N.
Departament de Genètica i
Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola, Spain. josune.jezenarro@gmail.com
Biosensors (Basel) 2020 Aug 20;10(9):102.
Abstract:
Legionella is a pathogenic bacterium, ubiquitous in freshwater environments
and able to colonise man-made water systems from which it can be transmitted to
humans during outbreaks. The prevention of such outbreaks requires a fast, low
cost, automated and often portable detection system. In this work, we present a
combination of sample concentration, immunoassay detection, and measurement by
chronoamperometry. A nitrocellulose microfiltration membrane is used as support
for both the water sample concentration and the Legionella immunodetection.
The horseradish peroxidase enzymatic label of the antibodies permits using the
redox substrate 3,3',5,5'-Tetramethylbenzidine to generate current changes
proportional to the bacterial concentration present in drinking water. Carbon
screen-printed electrodes are employed in the chronoamperometric measurements.
Our system reduces the detection time: from the 10 days required by the
conventional culture-based methods, to 2-3 h, which could be crucial to avoid
outbreaks. Additionally, the system shows a linear response (R2 value
of 0.99), being able to detect a range of Legionella concentrations
between 101 and 104 cfu·mL-1 with
a detection limit (LoD) of 4 cfu·mL-1.
Performance
of Legiolert Test vs. ISO 11731 to Confirm Legionella pneumophila Contamination
in Potable Water Samples
Scaturro M, Buffoni M,
Girolamo A, Cristino S, Girolamini L, Mazzotta M, Bucci Sabattini MA, Zaccaro
CM, Chetti L, Laboratory MAN, Bella A, Rota MC, Ricci ML.
Department of Infectious Diseases, Istituto Superiore di Sanità, Rome,
Italy. marialuisa.ricci@iss.it
Pathogens
2020 Aug 23;9(9):690.
Abstract:
Detection and enumeration of Legionella in
water samples is of great importance for risk assessment analysis. The plate
culture method is the gold standard, but has received several well-known
criticisms, which have induced researchers to develop alternative methods. The
purpose of this study was to compare Legionella counts obtained
by the analysis of potable water samples through the plate culture method and
through the IDEXX liquid culture Legiolert method. Legionella plate
culture, according to ISO 11731:1998, was performed using 1 L of water.
Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols.
Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them,
positive for L. pneumophila, serogroups 1 or 2-14 by plate culture,
were used for comparison with the Legiolert results. The Legiolert 10 mL test
detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37
positive samples, 27.6% and 30% respectively, out of the total samples analyzed.
No significant difference was found between either the Legiolert 10 mL and
Legiolert 100 mL vs. the plate culture (p = 0.9 and p =
0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests (p =
0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella
pneumophila detection and enumeration, as already shown in similar
studies. Like the plate culture method, the Legiolert assay is also suitable for
obtaining isolates for typing purposes, relevant for epidemiological
investigations.
Rapid
culture-based detection of Legionella
pneumophila using isothermal microcalorimetry with an improved evaluation
method
Fricke
C, Xu J, Jiang FL, Liu Y, Harms H, Maskow T.
Department
of Environmental Microbiology, Helmholtz‐Centre for Environmental Research – UFZ, Leipzig
Germany. Thomas.Maskow@ufz.de
Microb
Biotechnol 2020 Jul;13(4):1262-1272.
Abstract:
The detection and quantification of Legionella
pneumophila (responsible for legionnaire's disease) in water samples can be
achieved by various methods. However, the culture-based ISO 11731:2017, which is
based on counts of colony-forming units per ml (CFU·ml-1 ) is
still the gold standard for quantification of Legionella species (spp.). As a
powerful alternative, we propose real-time monitoring of the growth of L.
pneumophila using an isothermal microcalorimeter (IMC). Our results
demonstrate that, depending on the initial concentration of L.
pneumophila, detection times of 24-48 h can be reliably achieved. IMC may,
therefore, be used as an early warning system for L.
pneumophila contamination. By replacing only visual detection of growth by a
thermal sensor, but otherwise maintaining the standardized protocol of the ISO
11731:2017, the new procedure could easily be incorporated into existing
standards. The exact determination of the beginning of metabolic heat is often
very difficult because at the beginning of the calorimetric signal the thermal
stabilization and the metabolic heat development overlap. Here, we propose a new
data evaluation based on the first derivation of the heat flow signal. The
improved evaluation method can further reduce detection times and significantly
increase the reliability of the IMC approach.
Sensitivity
and Selectivity of Two Commercially Available Media for Legionella spp.
Recovery from Environmental Water Samples
Ditommaso S, Giacomuzzi M, Memoli G, Garlasco J, Zotti CM.
Department
of Public Health and Pediatrics, University of Turin, Turin, Italy. savina.ditommaso@unito.it
Pathogens
2020 Jun 29;9(7):523.
Abstract:
The quality control of culture media used for Legionella spp.
isolation and enumeration is paramount to achieve a satisfactory degree of
comparability among water testing results from different laboratories. Here, we
report on a comparative assessment of the sensitivity and selectivity of MWY and
BCYEα media supplied by two different manufacturers (i.e., Xebios
Diagnostics GmbH and Oxoid Ltd) for the detection of Legionella spp.
from environmental water samples. Even though our analysis showed an excellent
agreement between the recovery rates of the four media tested (90.5%), the
quantitative recovery of Legionella spp. colonies using Xebios
media was significantly greater than that achieved by Oxoid media (P =
0.0054). Furthermore, the sensitivity of detection was significantly higher when
samples were plated on MWY Xebios agar (P =
0.0442), while the selectivity of MWY appeared to be the same regardless of the
manufacturer. Furthermore, MWYXebios agar favored the growth of
much larger colonies compared to those observed on MWYOxoid agar.
Finally, MWYXebios medium enhanced the recovery of non-pneumophila
Legionella species. Collectively, our findings demonstrate that quality
control is crucial to ensure high selectivity and sensitivity of the culture
media used for the detection and enumeration of Legionella spp.
from environmental water resources. As water remediation measures strictly
depend on Legionella spp. recovery, culture protocol
standardization, as well as quality control of the culture media, is essential
to achieve intra- and interlaboratory reproducibility and accuracy.
Muyldermans
A, Descheemaeker P, Boel A, Desmet S, Van Gasse
N, Reynders M; National Expert Committee on Infectious Serology.
Department of Medical Microbiology, AZ
Sint-Jan Hospital, Bruges, Belgium. marijke.reynders@azsintjan.be.
Eur J Clin
Microbiol Infect Dis 2020
Apr;39(4):729-734.
Abstract: Currently, diagnosis of legionellosis
relies mainly on urinary antigen testing (UAT) for Legionella pneumophila
serogroup 1 (Lp1). However, this test has several limitations, particularly
missing non-Lp1 infections. The purpose of this large multicenter study was to
investigate the risk of missing legionellosis relying on UAT solely. Molecular
results of Legionella detection as part of a first line (syndromic)
testing algorithm for severe respiratory tract infections were investigated
retrospectively and compared with UAT results in 14 Belgian laboratories.
Overall, 44.4% (20/45) UAT results appeared false negative and were reclassified
as legionellosis based on PCR findings [Legionnaires' disease, 37.5% (15/40);
Pontiac fever, 100% (5/5)]. A total of 39.4% (26/66) diagnosis probably would
have been missed or delayed without a syndromic approach, as UAT or specific
molecular testing for Legionella was not requested by the clinician.
Furthermore, we confirmed the higher sensitivity of molecular Legionella
detection in lower respiratory tract compared with upper respiratory tract
specimens (p=0.010).
Serotyping and detection of pathogenecity loci
of environmental isolates of Legionella pneumophila using
MALDI-TOF MS
Kyritsi MA, Kristo I, Hadjichristodoulou
C.
Laboratory of Hygiene and
Epidemiology, School of Health Sciences, Department of Medicine, Larissa, Greece.
xhatzi@med.uth.gr
Int J Hyg Environ Health 2020 Mar;224:113441.
Abstract: BACKGROUND: The majority of Legionnaires' disease cases is
attributed to Legionella pneumophila serogroup 1 (Lp1).
Moreover, pathogenicity loci lvh and rtxA were associated with the ability of Lp
strains to cause the disease. Consequently, except from serogroup assignment the
detection of the aforementioned virulence genes during Legionella detection
in water samples, could help environmental risk assessment and the
implementation of targeted control measures. AIM: To establish
and validate a rapid and robust MALDI-TOF MS-based method for the assignment of
Lp isolates to serogroup and identify distinct peak biomarkers for the detection
of lvh and rtxA loci during environmental investigations. METHOD: Fifteen reference strains and 150 Lp environmental
isolates (70 Lp1 and 80 Lp2-15 strains) were used. All strains were PCR-tested
for the presence of lvh and rtxA loci. Independent training and validation
strain sets were constituted, and all strains were protein-extracted and
submitted to MALDI-TOF MS analysis. The raw spectra of the training set strains
obtained, were introduced into the Mass-Up software platform for biomarker
detection, for both serogroup assignment and pathogenicity loci detection.
Validation of the assigned biomarkers followed using the validation set strains. RESULTS: For serogroup assignment, the Mass-up analysis
indicated five potential discriminating peaks and correctly classified 115 out
of 132 validation set strains, displaying sensitivity of 87.5%, specificity of
86.7% and 87.1% accuracy. Concerning the pathogenicity loci detection, the
Mass-up analysis indicated two ion peaks for rtxA locus discrimination and one
peak for lvh locus discrimination. Concerning the lvh virulence gene, the
algorithm correctly classified 113 out of 137 positive and all negative strains
14 in total-showing sensitivity of 82.5%, specificity of 100% and 84.1% accuracy.
For rtxA locus, 134 out of 134 positive and 14 out of 17 negative strains were
correctly classified with sensitivity of 100%, specificity of 76.5% and 97.4%
accuracy. CONCLUSION: MALDI-TOF MS displayed good performance for Lp
serogroup assignment and detection of the lvh and rtxA virulence genes. These
findings could contribute to the rapid, inexpensive and comprehensive case
investigation and risk assessment. Further studies are needed to standardize and
evaluate the method using the direct target plate protein profiling instead of
protein extraction in order to simplify the protocol.
Badoux P, Kracht-Kosten L,
Herpers B, Euser S.
Regional Public Health Laboratory
Kennemerland, Haarlem, the Netherlands. p.badoux@streeklabhaarlem.nl
J Clin
Microbiol 2020
Feb;58(3):e01429-19.
Abstract: We
compared the clinical performance of the ImmuView L. pneumophila and L.
longbeachae Urinary Antigen test (SSI Diagnostica A/S, Hillerød,
Denmark) to the BinaxNOW Legionella Urinary Antigen Card (Binax,
Abbott, Lake Buff, IL) using urine specimens from patients suspected of having
pneumonia. In total, 100 frozen urine samples (derived from 50 Legionella cases
and 50 non-cases) were analyzed with both tests, as well as 200 non-frozen
prospectively collected samples. For urine samples of five Legionella cases
and two non-Legionella cases, the analytical sensitivity (limit of
detection) and repeatability were examined. The urine samples of the five Legionella cases
were diluted with urine samples that tested Legionella urinary
antigen negative with both tests. The analyses of the 100 frozen samples
resulted in a sensitivity and specificity of both the ImmuView and the BinaxNOW
of 96.0% (48/50) and 100% (50/50). Of the 200 non-frozen samples, there were
three samples that in both tests showed a positive result for L.
pneumophila The analyses of reproducibility showed that for the 34 (diluted)
samples that were tested at two consecutive times, 33 samples showed a
consistent result for both the ImmuView and the BinaxNOW test (Cohen's kappa's:
0.916 and 0.928). In addition, the ImmuView test may have detected two L.
longbeachae positive urine samples, although other diagnostic tests
could not confirm this. Both ImmuView and BinaxNOW showed high sensitivity and
specificity for the detection of L. pneumophila serogroup 1
antigen in urine samples from clinical patients with a suspected lower
respiratory tract infection.
Borella P, Vecchi E, Incerti F,
Marchesi I, Meacci M, Frezza G, Fregni Serpini G, Mansi A, Paduano S, Bargellini
A.
Department
of Biomedical, Metabolic and Neural Sciences, Section of Public Health,
University of Modena and Reggio Emilia, Modena, Italy. paola.borella@unimore.it
Int J Infect
Dis 2020 Feb;91:174-176.
Abstract: Three cases of pneumonia caused by Legionella
pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated
hospitalization were suspected as a healthcare-associated cluster. The
environmental investigation did not reveal the presence of legionellae in the
hospital patient rooms. Water samples collected from the homes of two patients
were also negative for Legionella spp. In the absence of environmental
strains potentially involved in the infections, we proceeded to genotype
environmental Lp1 strains isolated in the hospital during routine water sampling
during the decade 2009-2019 and recovered after long-term storage at -20°C.
These 'historical' strains exhibited a high grade of similarity and stability
over time, regardless of the disinfection systems. The different molecular
profiles shown among the clinical and environmental strains excluded a
nosocomial outbreak. The study suggests that the application of molecular typing
may be a useful tool to discriminate hospital vs community-acquired cases,
mostly for severely immunosuppressed patients in whom the symptomatology could
be insidious and the incubation period could be prolonged. Moreover, the
genotyping allowed us to exclude any link between the cases.
Isaac
TS, Sherchan SP.
Department of Global Environmental Health Sciences,
School of Public Health and Tropical Medicine, Tulane University, New Orleans,
LA, USA. sshercha@tulane.edu.
Environ Res Feb
2020;181:108847.
Abstract: Opportunistic premise plumbing pathogens
(OPPPs) in drinking water distribution systems are responsible for causing
numerous infections such as Legionnaires' disease and pneumonia through the
consumption of contaminated drinking water. The incidence of opportunistic
pathogens and the number of individuals at risk of contracting infections caused
by these OPPPs in drinking water has risen drastically in the past decade.
Pre-flush and post-flush water samples were collected from 64 houses in a rural
town in northeast Louisiana to determine drinking water quality in terms of
understanding abiotic and biotic factors on potential proliferation of OPPPs.
Physical and chemical water quality parameters, such as pH, temperature,
dissolved oxygen, salinity, and specific conductance were also measured. The
quantitative polymerase chain reaction (qPCR) results indicated that Legionella
spp. had the highest prevalence and was found in 46/64 samples (72%), followed
by Mycobacterium spp. which was found in 43/64 samples (67%), E. coli
in 31/64 samples (48%) and, Naegleria fowleri in 4/64 samples (6%)
respectively. The results indicate the persistence of Legionella spp. DNA
marker in these water samples.
Bayle
S, Martinez-Arribas B, Jarraud S, Giannoni P,
Garrelly L, Roig B, Cadière A.
EA7352 CHROME, University of Nîmes, Nîmes,
France. axelle.cadiere@unimes.fr
Heliyon 2020
Jan;6(1):e03149.
Abstract: Legionella risk assessment
is nowadays based on the presence and concentration of either Legionella
pneumophila or Legionella spp. Many species of Legionella can
cause Legionnaires' disease, indeed about half of the known species have been
associated with infection. The aim of this work was to develop a method to
assess the composition of the Legionella species community in
an environmental sample in order to have a better understanding of the
contamination of the ecosystem by pathogenic strains. The method is based on the
comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE
profiles of Legionella species. Such a database includes all
pathogenic Legionella strains. In order to homogenize and
normalize the different DGGE fingerprint, a reference marker has been built and
added during DGGE gel analysis. This study gives a valuable advance in the
methods available for the understanding of Legionella contamination
of water environments.
Reuter C, Slesiona N, Hentschel
S, Aehlig O, Breitenstein A, Csáki A, Henkel T, Fritzsche W.
Leibniz Institute of Photonic Technology
(Leibniz IPHT) Jena, Member of the Leibniz Research Alliance-Leibniz Health
Technologies, Jena, Germany. cornelia.reuter@leibniz-ipht.de
Appl Microbiol Biotechnol 2020
Jan;104(1):405-415.
Abstract: Recently Legionella
pneumophila is the main causative waterborne organism of severe respiratory
infections. Additionally, other Legionella species are documented as
human pathogens. In our work, we describe a rapid detection method which
combines two advantages for sensitive and specific detection of the genus Legionella:
the fast isothermal amplification method "Loop-mediated isothermal
AMPlification" (LAMP), and a colorimetric detection method using the metal
indicator hydroxynaphtol blue (HBN) which allows to determine an optical signal
with a simple readout (with the naked eye). Moreover, we present two approaches
for minimizing the assay volume using a stationary microchip LAMP and droplet
digital-based LAMP (ddLAMP) as promising highly sensitive setups.
Edagawa A, Kimura A, Miyamoto H.
Department of Environment Health, Osaka
Prefectural Institute of Public Health, Osaka, Japan. edagawa@iph.osaka.jp
Biocontrol
Sci 2019;24(4):213-220.
Abstract: We analyzed the contamination of
environmental water samples with Legionella spp. using a conventional
culture method, real-time quantitative PCR (qPCR), and real-time qPCR combined
with an amoebic co-culture method. Samples (n=110) were collected from 19
cooling towers, 31 amenity water facilities, and 60 river water sources of tap
water in Japan. Legionella was detected in only three samples (3/110,
2.7%) using the culture method. The rate of Legionella detection using
amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without
amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed
in 19 samples (19/110, 17.3%) using real-time qPCR subsequent to amoebic
co-culture, compared with identical samples analyzed without co-culture. Of
these 19 samples, 13 were identified as Legionella spp., including L.
pneumophila and L. anisa, and the non-culturable species were
identified as L. lytica and L. rowbothamii. This study showed that
the detection of Legionella spp., even in those samples where they were
not detected by the culture method, was possible using real-time qPCR and an
amoebic co-culture method. In addition, this analytical test combination is a
useful tool to detect viable and virulent Legionella spp.
Raphael
BH, Huynh T, Brown E, Smith JC, Ruberto I,
Getsinger L, White S, Winchell JM.
Respiratory Diseases Branch, CDC,
Atlanta, Georgia, USA. jwinchell@cdc.gov
mSphere 2019;4(1):e00649-18.
Abstract: Between 2000 and 2017, a total of 236 Legionella species
isolates from Arizona were submitted to the CDC for reference testing. Most of
these isolates were recovered from bronchoalveolar lavage specimens. Although
the incidence of legionellosis in Arizona is less than the overall U.S.
incidence, Arizona submits the largest number of isolates to the CDC for testing
compared to those from other states. In addition to a higher proportion of
culture confirmation of legionellosis cases in Arizona than in other states, all Legionella pneumophila isolates
are forwarded to the CDC for confirmatory testing. Compared to that from other
states, a higher proportion of isolates from Arizona were identified as
belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%).
Genome sequencing was conducted on 113 L. pneumophila clinical
isolates not known to be associated with outbreaks in order to understand the
genomic diversity of strains causing legionellosis in Arizona. Whole genome
multilocus sequence typing (wgMLST) revealed 17 clusters of isolates sharing at
least 99% identical allele content. Only two of these clusters contained
isolates from more than one individual with exposure at the same facility.
Additionally, wgMLST analysis revealed a group of 31 isolates predominantly
belonging to serogroup 6 and containing isolates from three separate clusters.
Single nucleotide polymorphism (SNP) and pangenome analysis were used to further
resolve genome sequences belonging to a subset of isolates. This study
demonstrates that culture of clinical specimens for Legionella spp.
reveals a highly diverse population of strains causing legionellosis in Arizona
which could be underappreciated using other diagnostic approaches.
IMPORTANCE: Culture of clinical specimens from patients with
Legionnaires' disease is rarely performed, restricting our understanding of the
diversity and ecology of Legionella Culture of Legionella from
patient specimens in Arizona revealed a greater proportion of non-serogroup 1 Legionella pneumophila isolates
than in other U.S. isolates examined. Disease caused by such isolates may go
undetected using other diagnostic methods. Moreover, genome sequence analysis
revealed that these isolates were genetically diverse and understanding these
populations may help in future environmental source attribution studies.
Dey R, Mount H, Ensminger AW,
Tyrrell GJ, Ward LP, Ashbolt NJ.
University of Alberta, Edmonton, Alberta, Canada. rafik@ualberta.ca
Emerg Infect
Dis 2019 Nov;25(11):2104-2107.
Abstract: Legionellosis was diagnosed in an
immunocompromised 3-year-old girl in Canada. We traced the source of the
bacterium through co-culture with an ameba collected from a hot tub in her home.
We identified Legionella pneumophila serogroup 6, sequence type 185, and
used whole-genome sequencing to confirm the environmental and clinical isolates
were of common origin.
Comparison of Whole-Genome
Sequences of Legionella pneumophila in Tap Water and in Clinical Strains,
Flint, Michigan, USA, 2016
Brown CL, Garner E,
Jospin G, Coil DA, Schwake DO, Eisen JA, Mukhopadhyay B, Pruden AJ.
Department
of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA, USA.
Emerg Infect Dis 2019
Nov;25(11):2013-2020.
Abstract: During the water crisis in Flint,
Michigan, USA (2014-2015), 2 outbreaks of Legionnaires' disease occurred in
Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella
pneumophila isolates submitted to a laboratory in Genesee County during the
second outbreak with 103 water isolates collected the following year. We
documented a genetically diverse range of L. pneumophila strains across
clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates,
3 water isolates from a Flint hospital, 1 water isolate from a Flint residence,
and the reference Paris strain) had a high degree of similarity (2-1,062
single-nucleotide polymorphisms), all L. pneumophila sequence type 1,
serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were
widespread in Flint hospital water samples but bore no resemblance to available
clinical isolates. L. pneumophila strains in Flint tap water after the
outbreaks were diverse and similar to some disease-causing strains.
Huang
Y, Ma Y, Miao Q, Pan J, Hu B, Gong Y, Lin
Y.
Department
of infectious diseases, Zhongshan Hospital, Fudan University, Shanghai, China. hu.bijie@zs-hospital.sh.cn
Ann Transl Med 2019 Oct;7(20):589.
Abstract: Legionella spp. is an
important pulmonary pathogen but rarely causes extra-pulmonary infections. We
report a case of joint infection caused by Legionella micdadei and Staphylococcus
aureus in a 54-year-old male with medication history of oral steroid
for systemic lupus erythematosus (SLE). He developed arthritis in his right
metacarpophalangeal (MCP) joints without precursor pneumonia. In the joint
aspirate, S. aureus was detected through culture. The existence
of L. micdadei and S. aureus were indicated by
metagenomic next-generation sequencing (mNGS) and confirmed by 16S rRNA sequence
analysis. After oral levofloxacin treatment for 54 days, the patient's symptoms
ameliorated and blood test results improved, which were consistent with the
dynamic trend of reads numbers in mNGS data. Our case included, arthritis caused
by Legionella spp. have been reported in 11 patients. However,
our case is the first to report septic arthritis caused by L. micdadei in
native joints and monitored by mNGS. This case demonstrated an application of
mNGS for etiological diagnosis and semi-quantification in joint aspirate. mNGS
may serve as a promising tool for rapid and accurate etiological diagnosis and
surveillance, contributing to appropriate antimicrobial drug applications and
timely medication adjustments when necessary.
Ginevra
C, Chastang J, David S, Mentasti M, Yakunin E,
Chalker VJ, Chalifa-Caspi V, Valinsky L, Jarraud S, Moran-Gilad J; ESCMID
Study Group for Legionella Infections (ESGLI).
ESCMID Study Group for Legionella Infections (ESGLI),
Basel, Switzerland; School of Public Health, Faculty of Health Sciences,
Ben-Gurion University of the Negev. Beer-Sheva, Israel; Public Health Services,
Ministry of Health, Jerusalem, Israel. giladko@post.bgu.ac.il
Clin Microbiol Infect 2019 Sep:S1198-743X(19)30487-2.
Abstract:
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.
Moreno Y, Moreno-Mesonero
L, García-Hernández J.
Research Institute of Water and Environmental
Ingeneering (IIAMA), Universitat Politècnica de València, Valencia, Spain. ymoren@upv.es
Environ Res 2019
Sep;176:108521.
Abstract: Despite all safety efforts, drinking and
wastewater can still be contaminated by Legionella and free-living
amoebae (FLA) since these microorganisms are capable of resisting disinfection
treatments. An amoebae cyst harboring pathogenic Legionella spp. can be a
transporter of this organism, protecting it and enhancing its infection
abilities. Therefore, the aim of this work is to identify by DVC-FISH viable Legionella
spp and Legionella pneumophila cells inside FLA from water sources in a
specific and rapid way with the aim of assessing the real risk of these waters.
A total of 55 water samples were processed, 30 reclaimed wastewater and 25
drinking water. FLA presence was detected in 52.7% of the total processed water
samples. When DVC-FISH technique was applied, the presence of viable
internalized Legionella spp. cells was identified in 69.0% of the total
FLA-positive samples, concretely in 70.0% and 66.7% of wastewater and drinking
water samples, respectively. L. pneumophila was simultaneously identified
in 48.3% of the total FLA-positive samples, specifically in 50.0% and 44.4% of
wastewater and drinking water samples, respectively. By culture, potentially
pathogenic Legionella cells were recovered in 27.6% of the total
FLA-positive bacteria, particularly in 35.0% and 11.1% of wastewater and
drinking water samples, respectively. These findings demonstrate that FLA may
promote resistance of bacteria to the performed disinfection treatments for
drinking as well as for wastewater. So, in addition to the risk for the presence
of pathogenic FLA in water it is necessary to take into account that these can
be transporters of the pathogenic bacteria Legionella, which are able to
survive inside them. The DVC-FISH method described here has been proved to be a
rapid and specific tool to identify pathogenic Legionella spp. and L. pneumophila
viable cells harboured by FLA in these water sources, posing particular
public health concern.
Moosavian M,
Moradzadeh M, Ghadiri A, Saki M.
Infectious and Tropical Diseases Research Center,
Health Research Institute, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran. mojtabamoosavian12@gmail.com
AIMS
Microbiol 2019 Aug;5(3):223-231.
Abstract: Legionella species are
widespread in natural water sources and man-made aqueous environments, as well
as freshwater. The present study was conducted owing to the lack of research
regarding the prevalence of Legionella spp in the water sources
of Ahvaz city in southwest Iran. In this study the macrophage infectivity
potentiator (mip) gene sequencing was used for identification of various Legionella species
isolated from different water sources. In this study, 144 water samples were
collected and inoculated on the buffered charcoal-yeast extract (BCYE) agar and
modified Wadowsky-Yee (MWY) medium. The DNA was extracted from positive cultures.
The Legionella species were confirmed by amplifying a 654 bp
fragment of the 16S rRNA gene. The mip gene of
all isolates were amplified by PCR and purified for sequencing. The mip gene
sequences were analyzed by jPHYDIT software version 1. The results showed a
13.9% (20/144) prevalence of Legionella spp. in water sources
of Ahvaz city, southwest Iran. Analyzing of the mip gene sequences showed, out
of 20 Legionella isolates, 13 isolates (54.1%) were positive
for L. pneumophila, 5 isolates (20.8%) were positive for L. worsleinsis,
one isolates for each one of L. dumoffi and L. fairfieldensis
(4.1%). According to our research, the occurrence of Legionella spp
in water sources could be a hazard for the health systems especially in the
hospitals. The regular monitoring of these water sources by health planners may
therefore be useful for decreasing the risk for Legionella spp.
infections.
Como J, Moffa MA, Bhanot N,
Min Z, Cole KS, Kuzyck J, Walsh TL.
Department of Medicine and Division of Infectious
Diseases, Allegheny General Hospital, Pittsburgh, PA, USA. James.Como@ahn.org
Eur J Clin
Microbiol Infect Dis 2019 Jul;38(7):1377-1382.
Abstract: The objective of this study was to
identify potential false-positive urine Legionella pneumophila (Legionella)
enzyme immunoassay test results. A total of 107 consecutive patients with
positive EIA tests were retrospectively analyzed over a 34-month period.
Concurrent blood, urine, and sputum cultures, as well as chest radiographic
findings, were reviewed in these patients. Twenty patients (19%) had no
radiographic evidence of pulmonary disease despite a positive EIA test. In those
20 patients, 14 also had growth of non-Legionella bacteria. Of patients
with an infiltrate or opacity on chest imaging, only 27 had Legionella sputum
cultures obtained, with Legionella culture growth occurring in 7 (26%).
Nine other patients had negative Legionella sputum cultures but the
growth of another pathogenic organism in blood, sputum, and/or urine cultures. Pseudomonas
aeruginosa was the most common organism isolated, found in 20% of patients
in the entire cohort. Twenty-five patients (23%) were characterized as having
probable false-positive Legionella urinary antigen EIA testing, and an
additional 17 patients (16%) were characterized as having possible
false-positive Legionella EIA tests. Our findings suggest that urine Legionella
EIA tests may lead to a substantial number of cases being misdiagnosed as
Legionnaires' disease in patients with non-Legionella bacterial
colonization or infection.
Congestrì F, Morotti M, Vicari R,
Pedna MF, Sparacino M, Torri A, Bertini S, Sambri V.
Unit of Microbiology, Greater Romagna Hub Laboratory,
Pievesestina di Cesena, Italy. francesco.congestri@auslromagna.it
J Clin Microbiol 2019
Jul;57(8):e00305-19.
Abstract:
Legionnaires' disease (LD) refers to a serious form of acute pneumonia caused by Legionella species.
LD can be difficult to diagnose because the signs and symptoms are nonspecific,
and therefore a rapid laboratory diagnosis is of paramount importance. In this
study, a recently introduced immunochromatographic test (Immunocatch Legionella;
Eiken Chemical Co., Ltd.) for Legionella pneumophila (serogroup
1) urinary antigen detection was compared with the Sofia Legionella fluorescent
immunoassay (FIA) (Quidel) (routinely used in our laboratory) and with the
widely used BinaxNOW Legionella assay (Alere). A total of 248 urine samples (60
frozen and 188 fresh) were evaluated. All of the samples were collected from
patients with high clinical suspicion of Legionnaires' disease. The three assays
were performed simultaneously according to the manufacturers' instructions. A
total of 180 concordant negative and 66 concordant positive results were
obtained. Only 2 discrepant results were registered. The sensitivity and
specificity of Immunocatch compared with Sofia were, respectively, 98.5% and
99.4%. Cohen's kappa coefficient and overall percent agreement between
Immunocatch and Sofia were also calculated and resulted in, respectively, 0.97
and 99.2%. These performances suggest that the Immunocatch test is a useful tool
for Legionella pneumophila (serogroup 1) urinary antigen
detection.
Barrette
I.
EnvironeX Group, 2325 Fernand-Lafontaine
Blvd, Longueuil, QC, Canada. isabellebarrette@labenvironex.com
J AOAC Int 2019
Jul;102(4):1235-1240.
Abstract: Background: Legionnaires'
disease is a potentially lethal pneumonia contracted through inhalation of
aerosolized water contaminated with Legionella bacteria.
Detection and control of L. pneumophila, the primary species
responsible for the disease, is critical to public health. In Québec, cooling
towers and evaporative condensers are required to follow a maintenance and
testing program to ensure L. pneumophila concentrations remain
at acceptable levels. Objective: This study compared a new
culture method based on the most probable number approach, Legiolert®,
with the formal culture method used at EnvironeX for regulatory compliance
testing to quantify L. pneumophila from cooling tower waters in
Québec. Methods: A split-sample analysis was performed in
which 401 samples from cooling towers in Québec were tested with both methods. Results: Results
with 74 positive samples showed that Legiolert provided a significant increase
in sensitivity for L. pneumophila compared with the agar plate
method. Cooling tower samples often contain non-Legionella flora
that necessitate multiple treatment and plating conditions to prevent
interference with the test. Legiolert showed little to no impact from non-Legionella organisms
in this study. Conclusions: Overall, Legiolert showed several
advantages over the agar plate method, including increased sensitivity, reduced
interference, a simplified test procedure, and an easy-to-read positive signal.
Miyashita N, Horita N, Higa F, Aoki Y, Kikuchi T, Seki M, Tateda K, Maki N, Uchino
K, Ogasawara K, Kiyota H, Watanabe A.
First Department of Internal Medicine, Division of
Respiratory Medicine, Infectious Disease and Allergology, Kansai Medical
University, Japan. miyashin@hirakata.kmu.ac.jp
J Infect
Chemother 2019 Jun;25(6):407-412.
Abstract: Background: Community-acquired
pneumonia (CAP) due to Legionella has a high mortality rate in patients
who do not receive adequate antibiotic therapy. In a previous study, we
developed a simple Legionella Score to distinguish patients with Legionella
and non-Legionella pneumonia based on clinical information at diagnosis.
In the present study, we validated this Legionella Score for the presumptive
diagnosis of Legionella CAP. Methods: This
validation cohort included 109 patients with Legionella CAP and 683
patients with non-Legionella CAP. The Legionella Score includes six
parameters by assigning one point for each of the following items: being male,
absence of cough, dyspnea, C-reactive protein (CRP) ≥18 mg/dL, lactate
dehydrogenase (LDH) ≥260 U/L, and sodium <134 mmol/L. Results: When
the Legionella CAP and non-Legionella CAP were compared by
univariate analysis, most of the evaluated symptoms and laboratory test results
differed substantially. The six parameters that were used for the Legionella
Score also indicated clear differences between the Legionella and non-Legionella
CAP. All Legionella patients had a score of 2 points or higher. The median
Legionella Scores were 4 in the Legionella CAP cases and 2 in the non-Legionella
CAP cases. A receiver operating characteristics curve showed that the area under
the curve was 0.93. The proposed best cutoff, total score ≥3, had
sensitivity of 93% and specificity of 75%. Conclusion: Our
Legionella Score was shown to have good diagnostic ability with a positive
likelihood of 3.7 and a negative likelihood of 0.10.
Mojtahedi
SY, Rahbarimanesh A, Noorbakhsh S, Shokri H,
Jamali-Moghadam-Siyahkali S, Izadi A.
Department of Pediatric Infectious
disease, Tehran University of Medical science, Tehran, Iran. dr.anahita.izadi@gmail.com
Eur J Transl
Myol, 2019 May;29(2):8120.
Abstract: Legionella pneumophila is
the causative agent of more than 95% cases of severe Legionella pneumonia.
Nosocomial pneumonias in different hospital wards is an important medical and
pharmaceutical concern. This study aimed to detect Legionella with
two methods: polymerase chain reaction (PCR) and detection of urine antigenic
test (UAT) in patients suffering from nosocomial pneumonia admitted to pediatric
intensive care unit (PICU) of children hospitals. This study was conducted in
PICU wards of Rasool Akram and Bahrami children hospitals, Tehran, Iran during
2013-2014. In patients diagnosed with hospital-acquired pneumonia, intratracheal
secretion samples for PCR and urine sample for UAT were taken. Simultaneously,
PCR and urinary antigen test were conducted using commercial kits. The results
of urinary antigen test and PCR were analyzed by SPSS v.19 for statistical
comparison. In this study, 96 patients aging 2.77 years on average with two age
peaks of less than 1 year and 7-8 year were enrolled. More than half of the
patients were under 1 year old. The most common underlying diseases were seizure,
Acute Lymphoblastic Lymphoma, Down syndrome and metabolic syndromes. The
positivity rate of Legionella urinary antigen test was 16.7% and positivity rate
of PCR test was 19.8%. There were no significant associations between the
results obtained by both assays with age, gender or underlying diseases. In
conclusion, PCR is a better detection method for Legionella infection
than urinary antigen test, but the difference between the two methods was not
significant.
Mobed
A, Hasanzadeh M, Babaie P, Agazadeh M,
Mokhtarzadeh A, Rezaee MA.
Drug Applied Research Center, Tabriz
University of Medical Sciences, Tabriz, Iran. hasanzadehm@tbzmed.ac.ir
Int J Biol
Macromol 2019 May;128:692-699.
Abstract: The specific diagnosis of hard-growing
bacteria is one of the most important concerns of medical bacteriology. Legionella
pneumophila is one of the most important bacteria in hard growth. In spite
remarkable trends in bacteriology, now day, culture is the gold standard for
detection of L. pneumophila. This work is an attempt to quantification of
L. pneumophila bacteria using a bioassay. The fabrication of a new
electrochemical DNA-based bioassay using gold nano architecture combined with as
a transducer substrate combined with toluidine blue (TB) as a redox marker was
performed successful. Also, the mixture of beta‑cyclodextrin and dopamine
as Poly (dopamine‑β‑Cyclodextrin)
was used to proper a biointerface for stabilization of gold nanoparticles
optimum immobilize of pDNA sequence (5-SH-TCGA TAC TCT CCC CGC CCC TT T
TGTATCGACG-3). So, a specific thiolated pDNA was immobilized on the transducer
substrate and DNA hybridization was followed by C-DNA sequence (5-ACA AAA GGG
GCG GGG AGA GTA-3) using square wave voltammetry and differential pulse
voltammetry. At the optimum conditions, linear range was 1 μM
to 1 ZM and low limit of quantification (LLOQ) was 1 Zepto-molar. L.
pneumophila were sensitively distinguished by the planned DNA sensor.
Finally, the engineered DNA based bioassay could be used for identifying the L.
pneumophila samples from patients or environments.
Ahmed
S, Liwak-Muir U, Walker D, Zoldowski A, Mears A,
Golovan S, Mohr S, Lem P, Harder C.
Spartan Bioscience Inc., 2934 Baseline
Road, Suite 500, Ottawa, ON, Canada. paul.lem@spartanbio.com.
J Water
Health 2019 Apr;17(2):237-253.
Abstract: Legionella pneumophila, found in
engineered water systems such as HVAC cooling towers, poses a significant public
health risk. Culture, though routinely used to quantify L. pneumophila,
has several disadvantages including long turnaround time, low sensitivity, and
inter-laboratory variability. In this study, we validated the performance of an
on-site quantitative polymerase chain reaction (qPCR) detection system for L.
pneumophila in accordance with International Standards Organization
Technical Specification 12869:2012. We evaluated specificity, limit of detection
and quantification, and calibration curve linearity. Additionally, we evaluated
whole system recovery and robustness using samples taken from taps and
evaporative cooling towers. We then compared the system's performance against
laboratory culture and laboratory qPCR across 53 cooling towers in a 12-week
in-field study. We found that concordance between on-site qPCR and culture was
both laboratory- and site/sample-dependent. Comparison of laboratory qPCR with
on-site qPCR revealed that laboratory results were highly variable and showed
little concordance. Some discordance may be explained by time delay between
sample collection and testing ('shipping effect') which may lead to inaccurate
reporting. Overall, our study highlights the value of on-site qPCR detection of L.
pneumophila, demonstrates that laboratories are prone to misreporting
results due to shipping effects, and reveals significant discordance between
laboratory qPCR and culture.
Moosavian
M, Seyed-Mohammadi S, Saki M, Shahi F,
Khoshkholgh Sima M, Afshar D, Barati S.
Department of Microbiology, Faculty of
Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. fatemehshahi87@gmail.com
Infect Drug Resist 2019
Mar;12:529-534.
Abstract: Background: Legionnaires'
disease is an important public health problem that can cause substantial
mortality and morbidity. Legionnaires' disease-risk estimation may be
compromised by uncertainties in Legionella-detection methods. The
aim of this study was the detection of L. pneumophila in
respiratory specimens of hospitalized patients with respiratory symptoms by
culture, PCR, and loop-mediated isothermal amplification (LAMP) methods. Methods: Sputum
and bronchoalveolar lavage samples were obtained from patients with pneumonia
admitted to teaching hospitals in Ahvaz, Iran from June 2016 to March 2017.
Isolation of Legionella spp. was done by culturing the samples
directly onto buffered charcoal-yeast extract and modified Wadowsky-Yee agar
medium. Then, PCR and LAMP assays were performed for detection of L.
pneumophila via its mip gene in respiratory specimens.
Results: A
total of 100 respiratory specimens were collected. Our results showed that 1% of
the samples were culture positive for Legionella spp., and 3%
and 7% of samples were positive for L. pneumophila using the mip gene
on PCR and LAMP assays, respectively.
Conclusion: Legionnaires'
disease should be considered in the diagnosis of pulmonary infectious diseases.
Also, the LAMP assay is a faster method with higher sensitivity and specificity
than conventional methods, such as PCR and culture, for laboratory diagnosis of
Legionnaires' disease.
Personnic N,
Striednig B, Hilbi H.
Institute of Medical Microbiology,
University of Zürich, Zürich, Switzerland. npersonnic@imm.uzh.ch
Methods Mol Biol 2019;1921:191-204.
Abstract: Legionella pneumophila resides in
multispecies biofilms, where it infects and replicates in environmental protozoa
such as Acanthamoeba castellanii. Studies on L. pneumophila
physiology and host-pathogen interactions are frequently conducted using clonal
bacterial populations and population level analysis, overlooking the remarkable
differences in single cell behavior. The fastidious nutrient requirements of
extracellular L. pneumophila and the extraordinary motility of Acanthamoeba
castellanii hamper an analysis at single cell resolution. In this chapter,
we describe a method to study L. pneumophila and its natural host A.
castellanii at single cell level by using an agarose embedment assay.
Agarose-embedded bacteria and infected cells can be monitored over several hours
up to several days. Using properly adapted flow chambers, agarose-embedded
specimens can be subjected to a wide range of fluctuating conditions.
Hochstrasser
R, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zürich, Switzerland. hilbi@imm.uzh.ch
Methods Mol
Biol 2019;1921:79-89.
Abstract:
The amoeba-resistant bacterium Legionella pneumophila infects humans
through aerosols and thereby can cause a life-threatening pneumonia termed
Legionnaires' disease. In the environment L. pneumophila forms and
colonizes biofilms, which usually comprise complex multispecies communities. In
these biofilms L. pneumophila persists and replicates intracellularly in
protozoa, such as the amoeba Acanthamoeba castellanii. The interactions
between sessile L. pneumophila in biofilms and their natural protozoan
hosts are not understood on a molecular level. Here, we describe a method to
visualize by confocal microscopy the formation and architecture of mono-species L.
pneumophila biofilms. Furthermore, we describe and quantify the migration or
"grazing" of A. castellanii in the biofilm. This allows
investigating on a molecular and cellular level L. pneumophila biofilm
formation and Legionella-amoeba interactions within biofilms.
Braun
RS, Mendis N, Li L, Faucher SP.
Faculty of Agricultural and Environmental Sciences,
Department of Natural Resource Sciences, McGill University, Ste-Anne-de-Bellevue,
QC, Canada. sebastien.faucher2@mcgill.ca
Methods Mol
Biol 2019;1921:45-53.
Abstract: Legionella pneumophila, among
other bacteria, may enter a viable but non-culturable state as a means for
survival in stressful conditions. Bacterial cells in the viable but
non-culturable state cannot grow on standard medium; however, they continue to
exhibit characteristics that are associated with live cells, such as respiration,
transcription, and cell wall integrity. The present paper outlines a detailed
protocol for the detection of viable but non-culturable L. pneumophila
cells via Syto® 9 and propidium iodide staining coupled with
flow cytometry.
Baume
M, Cariou A, Leveau A, Fessy N, Pastori F,
Jarraud S, Pierre S.
Hospices Civils de Lyon, Centre National
de Référence des Légionelles, Lyon, France. maud.baume@chu-lyon.fr
J Microbiol
Methods 2019 Feb;157:50-53.
Abstract: A value was assigned in 2009 to the Legionella
DNA Certified Reference Material, and the stability study conducted using
quantitative PCR found a low level of degradation. Herein, the Digital Droplet
PCR method for Legionella DNA was qualified and used to provide absolute
quantification of the CRM.
Kontchou
JA, Nocker A.
IWW Water Centre, Applied Microbiology, Mülheim an der
Ruhr, Germany.
J Microbiol
Methods 2019 Jan;156:68-76.
Abstract: Although a number of viability qPCR
assays have been reported to selectively detect signals from membrane-intact Legionella
pneumophila, the efficient suppression of amplification of DNA from dead
membrane-compromised bacteria remains an ongoing challenge. This research aimed
at establishing a new oligonucleotide combination that allows for a better
exclusion of dead Legionella pneumophila on basis of the mip gene.
Propidium monoazide (PMA) was chosen as viability dye. An oligonucleotide
combination for the amplification of a 633 bp sequence was established with 100%
specificity for different Legionella pneumophila strains compared with 17
other Legionella species tested. Apart from increasing amplicon length,
the study aimed at optimizing dye incubation time and temperature. An incubation
temperature of 45°C for 10 min was found optimal. Dye treatment of heat-killed
bacteria in the presence of EDTA improved signal suppression, whereas
deoxycholate also affected signals from live intact bacteria. Suppression of
signals from heat-treated bacteria was found to be approx. twice as efficient
compared to a commercial kit, although the detection sensitivity is superior
when targeting short amplicons. With a limit of detection of 10 genome copies
per PCR well and a 6-log signal reduction of bacteria killed at 80°C, the assay
appears useful for applications where pathogen numbers are not limiting and
where the priority is on the distinction between intact and damaged Legionella
pneumophila for the evaluation of hygienic risk and of disinfection
efficiency.
Quero S, Párraga-Niño N, Sabria
M, Barrabeig I, Sala MR, Jané M, Mateu L, Sopena N, Pedro-Botet ML,
Garcia-Nuñez M.
Infectious Diseases Unit, Hospital
Universitari Germans Trias i Pujol, Badalona, Spain. squero@igtp.cat e msabria.germantrias@gencat.cat
Sci Rep 2019
Jan;9(1):623.
Abstract: Legionnaires' disease (LD) is an
atypical pneumonia caused by the inhalation of Legionella. The methods
used for the diagnosis of LD are direct culture of respiratory samples and
urinary antigen detection. However, the sensitivity of culture is low, and the
urinary antigen test is specific only for L. pneumophila sg1. Moreover,
as no isolates are obtained, epidemiological studies cannot be performed. The
implementation of Nested-sequence-based typing (Nested-SBT) makes it possible to
carry out epidemiological studies while also confirming LD, especially in cases
caused by non-sg 1. Sixty-two respiratory samples from patients with Legionella
clinically confirmed by positive urinary antigen tests were cultured and tested
by Nested-SBT, following the European Study Group for Legionella Infections
(ESGLI) protocol. Only 2/62 (3.2%) respiratory samples were culture positive.
Amplification and sequencing of Nested-SBT genes were successfully performed in
57/62 samples (91.9%). The seven target genes were characterised in 39/57
(68.4%) respiratory samples, and the complete sequence type (ST) was obtained.
The mip gene was the most frequently amplified and sequenced. Nested-SBT is a
useful method for epidemiological studies in culture-negative samples, achieving
a 28.7-fold improvement over the results of culture studies and reducing the
time needed to obtain molecular epidemiological results.
Mobed
A, Hasanzadeh M, Agazadeh M, Mokhtarzadeh
A, Rezaee MA, Sadeghi J.
Drug Applied Research Center, Tabriz
University of Medical Sciences, Tabriz, Iran. hasanzadehm@tbzmed.ac.ir
Int J Biol
Macromol 2019 Jan;121:1295-1307.
Abstract: Fastidious bacteria are group of
bacteria that not only grow slowly but also have complex nutritional needs. In
this review, recent progress made on development of biosensing strategies
towards quantification of Legionella pneumophila as fastidious bacteria
in microbiology was investigated. In coincidence with medical bacteriology, it
is the most widely used biomonitoring, biosensors based on DNA and antibody.
Also, all of Legionella pneumophila genosensors and immunosensors that
developed in recent years were collected and analyzed. This review is meant to
provide an overview of the various types of bioassays have been developed for
determination of Legionella, along with significant advances over the
last several years in related technologies. In addition, this review described:
i) Most frequently applied principles in bioassay/biosensing of Legionella.
ii) The aspects of fabrication in the perspective of bioassay/biosensing
applications. iii) The potential of various electrochemical and optical bioassay/biosensing
for the determination of Legionella and the circumvention of the most
serious problem in immunosensing/immunoassay was discussed. iv) Some of bioassay/biosensing
has been discussed with and without labels. v) We also summarize the latest
developments in the applications of bioassay/biosensing methods for detection of
Legionella. vi) The development trends of optical and electrochemical
based bioassay/biosensing are also introduced.
Ricci ML, Grottola A, Fregni Serpini G, Bella A, Rota MC, Frascaro F, Pegoraro E,
Meacci M, Fabio A, Vecchi E, Girolamo A, Rumpianesi F, Pecorari M, Scaturro M.
Department of Infectious Diseases,
Istituto Superiore di Sanità, Rome, Italy. maria.scaturro@iss.it
Euro Surveill
2018 Dec;23(50):1800032.
Abstract:
Aim: To evaluate real-time PCR as a diagnostic method for Legionnaires' disease
(LD). Detection of Legionella DNA is among the laboratory
criteria of a probable LD case, according to the European Centre for Disease
Prevention and Control, although the utility and advantages, as compared to
culture, are widely recognised. Methods: Two independent laboratories, one using
an in-house and the other a commercial real-time PCR assay, analysed 354
respiratory samples from 311 patients hospitalised with pneumonia between
2010-15. The real-time PCR reliability was compared with that of culture and
urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive
and negative predictive values (PPV and NPV, respectively) were calculated.
Results: Overall PCR detected eight additional LD cases, six of which were due
to Legionella pneumophila (Lp) non-serogroup 1. The two
real-time PCR assays were concordant in 99.4% of the samples. Considering
in-house real-time PCR as the reference method, specificity of culture and UAT
was 100% and 97.9% (95% CI: 96.2-99.6), while the sensitivity was 63.6% (95%CI:
58.6-68.6) and 77.8% (95% CI: 72.9-82.7). PPV and NPV for culture were 100% and
93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4)
and 95.8% (95% CI: 93.5-98.2). Conclusion: Regardless of the real-time PCR assay
used, it was possible to diagnose LD cases with higher sensitivity than using
culture or UAT. These data encourage the adoption of PCR as routine laboratory
testing to diagnose LD and such methods should be eligible to define a confirmed
LD case.
Wells M, Lasek-Nesselquist E, Schoonmaker-Bopp D,
Baker D, Thompson L, Wroblewski D, Nazarian E, Lapierre P, Musser KA.
Wadsworth Center, New York State
Department of Health, Albany, NY, USA.
Erica.lasek-nesselquist@health.ny.gov
Infect Genet Evol 2018
Nov;65:200-209.
Abstract: We investigated the value of
whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses
in determining the relationships among and evolutionary rates of Legionella
species with long-term persistence in three healthcare facilities. We examined
retrospective clinical and environmental isolates of Legionella micdadei
and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA
fingerprints sampled over the course of up to 18 years. WGS analyses
demonstrated that heterogeneous populations of Legionella were present
within each facility despite displaying the same PFGE profiles. Additionally,
clustering of some clinical isolates with those from a separate but related
institution exposed a source of infection not previously detected, underscoring
the importance of considering phylogenetic relationships when assessing
epidemiological links. The data supported an average substitution rate of 0.80
SNPs per genome per year for L. micdadei but a reliable estimate for L.
pneumophila serogroup 1 could not be obtained due to complicating factors
such as non-chronological links among isolates and inadequate sampling depths.
While the substitution rate for L. micdadei is consistent with previous
estimates for L. pneumophila, the lack of a temporal signal in our
sequence data for L. pneuomphila serogroup 1 isolates suggests either
insufficient change to provide an estimate or variable evolutionary rates, which
could reflect the presence of both actively dividing and viable but
non-culturable Legionella spp. in the built environment. This study
highlights the increased discriminatory power of WGS SNP analysis as compared to
PFGE, emphasizes the need for extended sampling, and provides insight into the
evolution of Legionella from longitudinal investigations.
Párraga-Niño N, Quero S, Ventós-Alfonso
A, Uria N, Castillo-Fernandez O, Ezenarro JJ, Muñoz FX, Garcia-Nuñez M, Sabrià
M.
Unitat de
Malalties Infeccioses, Fundació Institut d'Investigació Germans Trias i Pujol,
Badalona, Spain. nparraga@igtp.cat
e msabria.germanstrias@gencat.cat
Talanta 2018
Nov;189:324-331.
Abstract: Waterborne pathogens are a global
concern for public health worldwide. Despite continuing efforts to maintain
water safety, water quality is still affected by deterioration and pollution. Legionella
pneumophila colonizes man-made water systems and can infect humans causing
Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health
issue and requires specific systems to control and detect these microorganisms.
Culture plate is the only technique currently approved but requires more than 10
days to obtain results. A rapid test that inform in hours about the presence of Legionella
pneumophila in water samples will improve the control of this pathogen
colonization. In order to control colonization by L. pneumophila we
developed a membrane filter method to capture and immunodetect this
microorganism in water samples. This membrane filter is used to retain the
bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently
we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking,
antibody incubation, washings and developing). On comparing our test with the
gold-standard, the most important finding is the considerably reduction in time
maintaining the same detection limit. This rapid test is easily automated for L.
pneumophila detection allowing a comprehensive surveillance of L.
pneumophila in water facilities and reducing the variability in the analyses
due to the low need for manipulation. Moreover, corrective measures may be
applied the same day of the analysis. This method considerably reduces the
detection time compared with the conventional, gold-standard detection culture
method that requires more than 10 days, being decisive to prevent outbreaks.
Nuthong
B, Wilailuckana C, Tavichakorntrakool R, Boonsiri
P, Daduang S, Bunyaraksyotin G, Suphan O, Daduang J.
Centre for Research and Development of
Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon
Kaen University, Khon Kaen, Thailand. jurpoo@kku.ac.th
J Appl
Microbiol 2018 Nov;125(5):1534-1540.
Abstract: Aims: To
develop and evaluate a DNA-gold nanoparticle (DNA-AuNP) probe assay to detect Legionella
pneumophila, which causes Legionnaires' disease, compared with the gold
standard culture method. Methods and
results: Gold
nanoparticles (AuNPs) were conjugated with DNA probes to detect the mip gene of L.
pneumophila. The DNA-AuNP probe assay was evaluated for its specificity,
sensitivity and stability. The results showed that only L. pneumophila
mixed with this probe resulted in a red solution that was easily detected by the
naked eye, and the colour was stable when 10 mmol l-1 MgSO4 was
added. The 100 Legionella isolates and 10 other bacteria led to 100%
specificity. Compared with the culture method, our method showed a 100% negative
predictive value, 100% sensitivity (kappa=0.87), and a detection limit of 4.5 ng
DNA μl-1 with
a 6-min response time for the 124 colonies suspected of being Legionella.
The DNA-AuNP probe reagents were stable for more than 6 months. Conclusions: The
developed DNA-AuNP probe assay has good negative predictive value, sensitivity,
rapidity and ease of use, which is helpful for ruling out negative samples. Significance and impact of the study: The
DNA-AuNP probe assay can detect the mip gene of L. pneumophila. Therefore,
it may be an alternative method for screening colonies suspected of being L.
pneumophila.
Boss R, Baumgartner A, Kroos
S, Blattner M, Fretz R, Moor D.
Federal Food Safety and Veterinary
Office, Berne, Switzerland.
renate.boss@blv.admin.ch
e dominik.moor@blv.admin.ch
Appl Microbiol 2018 Oct;125(4):1216-1225.
Abstract:
Aims: A molecular
method for a rapid detection of viable Legionella pneumophila of all
serogroups in tap water samples was developed as an alternative to the reference
method (ISO). Legionellae are responsible for Legionnaires' disease, a severe
pneumonia in humans with high lethality. Methods and
results: The
developed method is based on a nutritional stimulation and detection of an
increase in precursor 16S rRNA as an indicator for viability. For quantification,
DNA was detected by qPCR. This method was compared to the ISO method using water
samples obtained from public sports facilities in Switzerland. The sensitivity
and specificity were 91 and 97%, respectively, when testing samples for
compliance with a microbiological criterion of 1000 cell equivalents per l. Conclusion: The
new method is sensitive and specific for L. pneumophila and allows
results to be obtained within 8 h upon arrival, compared to one week or more by
the ISO method. Significance and
impact of the study: The method
represents a useful tool for a rapid detection of viable L. pneumophila
of all serogroups in water by molecular biology. It can be used as an
alternative to the ISO method for official water analysis for legionellae and
particularly when a short test time is required.
Graells T, Hernández-García M,
Pérez-Jové J, Guy L, Padilla E.
Departament de Microbiologia, CATLAB Centre Analítiques
AIE, Viladecavalls, Barcelona, Spain. tiscar.graells@e-campus.uab.cat
Environ Res 2018 Oct:166:638-646.
Abstract:
Objectives: The
aim of this study was to monitor the spread, persistence and antibiotic
resistance patterns of Legionella spp. strains found in a hospital water
distribution system. These environmental studies are intended to help detect the
presence of antibiotic resistant strains before they infect patients. Methods: Antimicrobial
surveillance tests were performed at 27 different sampling points of the water
network of a large Spanish hospital over two years. Water samples were screened
for Legionella according to ISO 11731:2007. Legionella spp.
isolates were identified by serotyping and by mass spectrometry (MALDI-ToF).
Epidemiological molecular typing was done by Pulse-Field Gel Electrophoresis (PFGE)
and by Sequence-Based Typing (SBT). Antibiotic susceptibility tests were
performed using disk diffusion and ETEST®. Results: Legionella
spp. were recurrently isolated for 2 years. All isolates belonged the same group,
L. pneumophila serogroups 2-14. Isolates were all attributed by SBT to
sequence type (ST) ST328, although PFGE revealed 5 different patterns. No
significant change in antibiotic susceptibility could be observed for this study
period, irrespectively of the method used. Conclusion: Colonization
of water systems by Legionella spp. is still occurring, although all the
prevention rules were strictly followed. Antibiotic resistance monitoring may
help us to find resistance in bacteria with environmental reservoirs but
difficult to isolate from patients. The knowledge of the antibiotic
susceptibility in environmental strains may help us to predict changes in
clinical strains. This study might also help reconsidering Legionnaires' disease
(LD) diagnostic methods. L. pneumophila serogroups 2-14 present all along
the time of the investigation in the water distribution system can cause LD.
However, they may not be detected by routine urine tests run on patients,
thereby missing an ongoing LD infection.
Rech MM, Swalla BM, Dobranic JK.
EMSL Analytical, Inc., 1010 Yuma Street, Denver, CO,
80204, USA. mrech@emsl.com
Curr
Microbiol 2018 Oct;75(10):1282-1289.
Abstract: Legiolert® is a new culture method for
quantification of Legionella pneumophila, which is the primary species
associated with Legionnaires' disease. The test is based on a most probable
number approach and differs significantly from traditional culture methods by
providing results at 7 days, rapid sample preparation and analysis, and
objective interpretation of test results. In this study, we compared the
performance of Legiolert with the U.S. Centers for Disease Control and
Prevention (CDC) method for detection of L. pneumophila from non-potable
samples, primarily comprising cooling tower waters. Our results demonstrated no
significant difference between Legiolert and the CDC method for quantification
of L. pneumophila. However, Legiolert showed a significant increase in
sensitivity when water samples containing higher L. pneumophila
concentrations were examined. Cooling tower waters often contain non-Legionella
organisms (NLO) that interfere with traditional Legionella test methods,
and we observed varying degrees of NLO interference on many CDC method plates.
In contrast, Legiolert was resistant to NLO interference and produced a very low
rate of false-positive results. Collectively, Legiolert is a sensitive and
specific method for quantification of L. pneumophila from non-potable
water that provides advantages over the CDC method.
David S, Mentasti M, Parkhill
J, Chalker VJ.
Centre
for Genomic Pathogen Surveillance, Wellcome Genome Campus, Hinxton, Cambridge,
United Kingdom. sd12@sanger.ac.uk
Clin Microbiol Infect 2018 Sep;24(9):1020.e1-1020.e4.
Abstract: Objectives: Legionella
pneumophila is the leading cause of Legionnaires' disease, a severe form of
pneumonia acquired from environmental sources. Investigations of both sporadic
cases and outbreaks rely mostly on analysis of a single to a few colony pick(s)
isolated from each patient. However, because of the lack of data describing
diversity within single patients, the optimal number of picks is unknown. Here,
we investigated diversity within individual patients using sequence-based typing
(SBT) and whole-genome sequencing (WGS). Methods: Ten
isolates of L. pneumophila were obtained from each of ten
epidemiologically unrelated patients. SBT and WGS were undertaken, and
single-nucleotide polymorphisms (SNPs) were identified between isolates from the
same patient. Results: The
same sequence type (ST) was obtained for each set of ten isolates. Using genomic
analysis, zero SNPs were identified between isolates from seven patients, a
maximum of one SNP was found between isolates from two patients, and a maximum
of two SNPs was found amongst isolates from one patient. Assuming that the full
within-host diversity has been captured with ten isolates, statistical analyses
showed that, on average, analysis of one isolate would yield a 70% chance of
capturing all observed genotypes, and seven isolates would yield a 90% chance. Conclusions: SBT
and WGS analyses of multiple colony picks obtained from ten patients showed no,
or very low, within-host genomic diversity in L. pneumophila, suggesting
that analysis of one colony pick per patient will often be sufficient to obtain
reliable typing data to aid investigation of cases of Legionnaires' disease.
Helmi K, David F, Di Martino P,
Jaffrezic MP, Ingrand V.
Veolia Recherche et Innovation, Chemin de la Digue,
Maisons-Laffitte, France. karim.helmi@veolia.com
J Microbiol
Methods 2018 Sep;152:201-209.
Abstract: The control of Legionella
proliferation in cooling tower water circuits requires regular monitoring of
water contamination and effective disinfection procedures. In this study, flow
cytometry was assessed to monitor water contamination and disinfection treatment
efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II),
cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome
V6). A total of 27 cooling tower water samples were analyzed in order to assess
water contamination levels regarding viable populations: standard culture, ATP
measurement and flow cytometry methods were compared. Flow cytometry and plate
counts methods showed a significant correlation for changes in concentrations
despite a 1 to 2-log difference regarding absolute quantification. Concerning
intracellular activity, the use of two different flow cytometers (FACSCanto™
II and Accuri™ C6) showed no statistical difference while a difference was
observed between flow cytometry and usual methods (culture and ATP measurement).
The standard culture and flow cytometry methods were also compared for in vitro
bacteria inactivation measurements in the presence of 3 different types of
oxidizing biocides commonly used for cooling tower disinfection. Reductions
observed ranged between 1 and 2 log depending on (1) the detection method, (2)
the bacterial population origin and/or (3) the active biocide molecule used. In
conclusion, flow cytometry represents an efficient, accurate and fast approach
to monitor water contamination and biocide treatment efficiency in cooling
towers.
Division of Pulmonology, Department of
Internal Medicine, Soonchunhyang University College of Medicine, Cheonan, Korea.
rk.ca.cmhcs@ankouj
Korean J Intern Med 2018 Sep;33(5):941-951.
Abstract:
Background/aims: To
use serological and multiplex polymerase chain reaction (PCR) assays to examine
sputum samples from patients experiencing acute exacerbation of chronic
obstructive pulmonary disease (AECOPD) for the presence of atypical pathogens,
including Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella
pneumophila. Methods: From
September 2012 to February 2014, 341 patients with AECOPD attending outpatient
clinics were enrolled as part of a randomized, double-blind, multicenter study.
A commercial enzyme-linked immunosorbent assay was used to measure serum
immunoglobulin M (IgM) and IgG antibody titers on the first day of the study and
at 36 days post-enrollment. Multiplex PCR was used to test sputum samples for
the presence of atypical pathogens. A urinary antigen test for L. pneumophila
was performed on the first day. Results: Nineteen
patients (5.6%) showed serological evidence of acute infection with M.
pneumoniae. Also, one and seven patients (2%) showed serological evidence of
acute infection with C. pneumoniae and L. pneumophila,
respectively. All DNA samples were negative for M. pneumoniae, C.
pneumoniae, and L. pneumophila according to PCR. Only one urine
sample was positive for L. pneumophila antigen, but serologic evidence
was lacking. Conclusion: Serological
testing suggested that infection by atypical pathogens during AECOPD was
relatively uncommon. In addition, PCR provided no direct evidence of infection
by atypical pathogens. Thus, atypical pathogens may not be a major cause of
AECOPD in South Korea.
Spies K, Pleischl S, Lange B,
Langer B, Hübner I, Jurzik L, Luden K, Exner M.
Universitätsklinikum Bonn, Institut für Hygiene und
Öffentliche Gesundheit, Bonn, Germany. kirsten.spies@ukbonn.de
Int J Hyg
Environ Health 2018 Aug;221(7):1047-1053.
Abstract: Due to the promising results of a
previous study of the performance of the novel MPN method (Legiolert™/Quanti-Tray®)
compared to ISO 11731-2, this study was performed to compare Legiolert for Legionella
pneumophila with the German regulatory requirements methods ISO 11731-2 (100
ml membrane filtration) and ISO 11731 (1 ml direct plating) for the enumeration
of L. pneumophila and Legionella spp. from potable water. Data
from a multi-laboratory study according to ISO 17994 showed that Legiolert
yielded on average higher counts of L. pneumophila than the ISO 11731-2
method, but the comparison with ISO 11731 was inconclusive due to the number of
samples needing to be tested. Likewise, comparisons of the MPN method for 100 ml
to the highest result of either ISO 11731 or ISO 11731-2 according to Federal
Environmental Agency recommendation (2012) yielded no conclusive difference,
regardless of whether non-pneumophila species of Legionella were
included in the evaluation. The MPN method has a high specificity for L.
pneumophila of 97.9% which compares favourably to the specificity of 95.3%
quoted for ISO 11731. The new method represents a significant improvement in the
enumeration of L. pneumophila from drinking water and related samples.
Fleres G, Couto N, Lokate M, van
der Sluis LWM, Ginevra C, Jarraud S, Deurenberg RH, Rossen JW, García-Cobos S,
Friedrich AW.
Department of Medical Microbiology,
University Medical Center Groningen, University of Groningen, Groningen, The
Netherlands. g.fleres@umcg.nl
Microorganisms 2018
Jul;6(3):71.
Abstract: This study aims to assess contamination
with Legionella spp. in water from dental chair units (DCUs) of
a hospital dental ward and to perform its molecular characterization by
whole-genome sequencing (WGS). We collect eight water samples (250 mL) from four
DCUs (sink and water-syringe). Samples are tested for the presence of Legionella spp.
(CFUs/mL) by culturing according to the Nederland Norm (NEN) 6265. Three DCUs
are found positive for Legionella anisa, and four isolates are
cultured (sink n=2, water-syringe n=1; two isolates from
the same chair) with 1×10² CFU/mL. Whole-genome multi-locus sequence typing (wgMLST)
results indicate that all strains belong to the same cluster with two to four
allele differences. Classical culture combined with WGS allows the
identification of a unique clone of L. anisa in several DCUs in
the same hospital dental ward. This may indicate a common contamination source
in the dental unit waterlines, which was fixed by replacing the chairs and main
pipeline of the unit. Our results reveal tap water contamination in direct
contact with patients and the usefulness of WGS to investigate bacterial
molecular epidemiology.
Borthong J,
Omori R, Sugimoto C, Suthienkul O, Nakao R, Ito K.
Division of Bioinformatics, Research Center for
Zoonosis Control, Hokkaido University, Sapporo, Japan. itok@czc.hokudai.ac.jp
Front
Microbiol 2018 Jun;9:1272.
Abstract: Metagenomic analysis has become a
powerful tool to analyze bacterial communities in environmental samples. However,
the detection of a specific bacterial species using metagenomic analysis remains
difficult due to false positive detections of sequences shared between different
bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic
analyses were conducted on samples collected along a stream and ponds in the
campus of Hokkaido University. We compared different database search methods for
bacterial detection by focusing on Legionella pneumophila. In this
study, we used L. pneumophila-specific nested PCR as a gold standard
to evaluate the results of the metagenomic analysis. Comparison with the results
from L. pneumophila-specific nested PCR indicated that a blastn
search of shotgun reads against the NCBI-NT database led to false positive
results and had problems with specificity. We also found that a blastn search of
shotgun reads against a database of the catalase-peroxidase (katB) gene
detected L. pneumophila with the highest area under the
receiver operating characteristic curve among the tested search methods;
indicating that a blastn search against the katB gene database
had better diagnostic ability than searches against other databases. Our results
suggest that sequence searches targeting long genes specifically associated with
the bacterial species of interest is a prerequisite to detecting the bacterial
species in environmental samples using metagenomic analyses.
Welin
A, Weber S, Hilbi H.
Institute of Medical Microbiology, University of Zürich,
Zürich, Switzerland. hilbi@imm.uzh.ch
Appl Environ
Microbiol 2018 May;84(11):e00158-18.
Abstract: The ubiquitous environmental bacterium Legionella
pneumophila survives and replicates within amoebae and human
macrophages by forming a Legionella-containing vacuole (LCV). In an
intricate process governed by the bacterial Icm/Dot type IV secretion system and
a plethora of effector proteins, the nascent LCV interferes with a number of
intracellular trafficking pathways, including retrograde transport from
endosomes to the Golgi apparatus. Conserved retrograde trafficking components,
such as the retromer coat complex or the phosphoinositide (PI) 5-phosphatase D.
discoideum 5-phosphatase 4 (Dd5P4)/oculocerebrorenal syndrome of Lowe (OCRL),
restrict intracellular replication of L. pneumophila by an
unknown mechanism. Here, we established an imaging flow cytometry (IFC) approach
to assess in a rapid, unbiased, and large-scale quantitative manner the role of
retrograde-linked PI metabolism and actin dynamics in the LCV composition.
Exploiting Dictyostelium discoideum genetics, we found that
Dd5P4 modulates the acquisition of fluorescently labeled LCV markers, such as
calnexin, the small GTPase Rab1 (but not Rab7 and Rab8), and retrograde
trafficking components (Vps5, Vps26, Vps35). The actin-nucleating protein and
retromer interactor WASH (Wiskott-Aldrich syndrome protein [WASP] and suppressor
of cAMP receptor [SCAR] homologue) promotes the accumulation of Rab1 and Rab8 on
LCVs. Collectively, our findings validate IFC for the quantitative and unbiased
analysis of the pathogen vacuole composition and reveal the impact of
retrograde-linked PI metabolism and actin dynamics on the LCV composition. The
IFC approach employed here can be adapted for a molecular analysis of the
pathogen vacuole composition of other amoeba-resistant pathogens. IMPORTANCE:
Legionella pneumophila is an amoeba-resistant environmental
bacterium which can cause a life-threatening pneumonia termed Legionnaires'
disease. In order to replicate intracellularly, the opportunistic pathogen forms
a protective compartment, the Legionella-containing vacuole (LCV).
An in-depth analysis of the LCV composition and the complex process of pathogen
vacuole formation is crucial for understanding the virulence of L.
pneumophila. Here, we established an imaging flow cytometry (IFC) approach
to assess in a rapid, unbiased, and quantitative manner the accumulation of
fluorescently labeled markers and probes on LCVs. Using IFC and L.
pneumophila-infected Dictyostelium discoideum or defined
mutant amoebae, a role for phosphoinositide (PI) metabolism, retrograde
trafficking, and the actin cytoskeleton in the LCV composition was revealed. In
principle, the powerful IFC approach can be used to analyze the molecular
composition of any cellular compartment harboring bacterial pathogens.
Descours G,
Hannetel H, Reynaud JV, Ranc AG, Beraud L, Kolenda C, Campese C, Lina G, Ginevra
C, Jarraud S.
Hospices Civils de Lyon, Groupement Hospitalier Nord,
National Reference Centre for Legionella, Institute for Infectious Agents, Lyon,
France. ghislaine.descours@univ-lyon1.fr
J Clin Microbiol 2018 Apr;56(5):e01361-17.
Abstract: 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.
Kozak-Muiznieks
NA, Morrison SS, Mercante JW, Ishaq MK, Johnson
T, Caravas J, Lucas CE, Brown E, Raphael BH, Winchell JM.
Division of Bacterial Diseases, National Center for
Immunization and Respiratory Diseases, CDC, Atlanta, GA, USA. Jwinchell@cdc.gov
Infect Genet
Evol 2018 Apr;59:172-185.
Abstract: The majority of Legionnaires' disease (LD)
cases are caused by Legionella pneumophila, a genetically heterogeneous
species composed of at least 17 serogroups. Previously, it was demonstrated that
L. pneumophila consists of three subspecies: pneumophila, fraseri
and pascullei. During an LD outbreak investigation in 2012, we detected
that representatives of both subspecies fraseri and pascullei
colonized the same water system and that the outbreak-causing strain was a new
member of the least represented subspecies pascullei. We used partial
sequence-based typing consensus patterns to mine an international database for
additional representatives of fraseri and pascullei subspecies. As
a result, we identified 46 sequence types (STs) belonging to subspecies fraseri
and two STs belonging to subspecies pascullei. Moreover, a recent
retrospective whole genome sequencing analysis of isolates from New York State
LD clusters revealed the presence of a fourth L. pneumophila subspecies
that we have termed raphaeli. This subspecies consists of 15 STs.
Comparative analysis was conducted using the genomes of multiple members of all
four L. pneumophila subspecies. Whereas each subspecies forms a distinct
phylogenetic clade within the L. pneumophila species, they share more
average nucleotide identity with each other than with other Legionella
species. Unique genes for each subspecies were identified and could be used for
rapid subspecies detection. Improved taxonomic classification of L.
pneumophila strains may help identify environmental niches and virulence
attributes associated with these genetically distinct subspecies.
Wagner
K, Springer B, Imkamp F, Opota O, Greub G, Keller
PM.
Institute of Medical Microbiology, University of Zurich,
Switzerland. pkeller@imm.uzh
Int J Med
Microbiol 2018 Apr;308(3):317-323.
Abstract: Pneumonia is a severe infectious disease.
In addition to common viruses and bacterial pathogens (e.g. Streptococcus
pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae,
Mycoplasma pneumoniae and Legionella spp. can cause severe
atypical pneumonia. They do not respond to penicillin derivatives, which may
cause failure of antibiotic empirical therapy. The same applies for infections
with B. pertussis and B. parapertussis, the cause of pertussis
disease, that may present atypically and need to be treated with macrolides.
Moreover, these fastidious bacteria are difficult to identify by culture or
serology, and therefore often remain undetected. Thus, rapid and accurate
identification of bacterial pathogens causing atypical pneumonia is crucial. We
performed a retrospective method evaluation study to evaluate the diagnostic
performance of the new, commercially available Lightmix® multiplex
RT-PCR assay that detects these fastidious bacterial pathogens causing atypical
pneumonia. In this retrospective study, 368 clinical respiratory specimens,
obtained from patients suffering from atypical pneumonia that have been tested
negative for the presence of common agents of pneumonia by culture and viral PCR,
were investigated. These clinical specimens have been previously characterized
by singleplex RT-PCR assays in our diagnostic laboratory and were used to
evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR.
The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies
for different in-panel organisms and showed identical performance
characteristics with respect to specificity and sensitivity as in-house
singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex
RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool
with high throughput potential. The time-to-result using an automated DNA
extraction device for respiratory specimens followed by multiplex RT-PCR
detection was below 4 h, which is expected to significantly improve diagnostics
for atypical pneumonia-associated bacterial pathogens.
Moriguchi
S, Abe M, Kimura M, Yoshino C, Baba M,
Okada C, Izutsu K, Taniguchi S, Araoka H, Yoneyama A.
Department of Hematology, Toranomon Hospital, Japan. muneyoshi-k@toranomon.gr.jp
Intern Med 2018
Apr;57(7):1045-1048.
Abstract: A 60-year-old man developed pneumonia
after undergoing autologous peripheral blood stem cell transplantation for
diffuse large-B cell lymphoma. A urinary antigen test and sputum culture were
both negative for Legionella pneumophila; however, a sputum sample that
was examined by loop-mediated isothermal amplification (LAMP) was positive for Legionella
spp. On admission, the results of blood culturing using a BACTEC system were
negative for 7 days. However, L. pneumophila serogroup 5 was detected in
a blood subculture using WYOα
medium. The patient was successfully treated with a fluoroquinolone-based
regimen. LAMP is useful for the diagnosis of Legionella spp.
Dilger T, Melzl H, Gessner A.
AGROLAB Labor GmbH, Eching am Ammersee, Germany. thorsten.dilger@agrolab.de
Int J Hyg
Environ Health 2018 Mar;221(2):199-210.
Abstract: Legionellae constitute a frequent
contamination of warm water systems and can lead to serious infections.
Therefore, in many countries it is mandatory to monitor warm water systems for
their presence. The method of examination in Germany is regulated by guideline
ISO 11731 and DIN EN ISO 11731-2, and the results are reported as concentration
of Legionella spp. Only limited information is available on the presence of
individual species of Legionellae in the examined systems, since most
investigations and research focus solely on Legionella pneumophila as the
most important human pathogen. In this study 76,220 samples obtained from 13,397
warm water systems originating from 24 different zip code districts covering an
area of more than 71,000km2 in southern Germany were examined.
This resulted in the identification of 47,924 Legionella isolates to the
species level using a MALDI-TOF mass spectrometry-based method. Legionella
species distribution was analyzed with respect to warm water system type,
geographic region (defined as zip code district) and temperature during sample
taking. Overall, 20.7% of the samples were found positive for Legionella
species and 14 different species of Legionella were recovered. These were
not equally present throughout the geographic area investigated, but instead an
individual regional diversity of Legionella species was observed for the
examined zip code districts. Although Legionella pneumophila represented
84% of all contaminations found, depending on the geographical region its
proportion varied substantially between 57.5% and 91.2%. The occurrence of other
species was also of importance since they accounted for up to 42% of
contaminations regionally, with Legionella londiniensis being most
prominent representing up to 38.8% of recovered colonies. In addition, the
influence of temperature on the individual species was disparate, but the
temperature range between 50°C and 59°C was identified as the optimal
condition for facilitating emergence of the majority of recovered Legionella
species. The identification of Legionella to the species level by
MALDI-TOF allowed for a more concise depiction of the regional distribution and
the ecology of this genus and may be of additional value when counter measures
need to be initiated.
Petrisek R,
Hall J.
H.P. Environmental, Inc., 104 Elden Street, Herndon,
VA, USA. rpetrisek@hpenviron.com
J Water
Health 2018 Feb;16(1):25-33.
Abstract:
This study compares the performance of a novel most
probable number (MPN) method (Legiolert™/Quanti-Tray®) with
Standard Methods for the Examination of Water and Wastewater 9260 J for the
enumeration of Legionella pneumophila from potable and nonpotable waters.
Data from the study showed that Legiolert exhibited higher sensitivity for the
detection of L. pneumophila for potable water and equivalent sensitivity
for nonpotable water. The Legiolert medium had a high specificity with no false
positive signals reported for either water type. The new method represents a
significant improvement in usability and accuracy in the enumeration of L.
pneumophila.
Zhan XY, Zhu QY.
Guangzhou KingMed Center for Clinical Laboratory,
Guangzhou, China. tsinghan@126.com
PLoS One 2018
Feb;13(2):e0190986.
Abstract: Inadequate discriminatory power to
distinguish between L. pneumophila isolates, especially those belonging
to disease-related prevalent sequence types (STs) such as ST1, ST36 and ST47, is
an issue of SBT scheme. In this study, we developed a multilocus sequence typing
(MLST) scheme based on two non-virulence loci (trpA, cca) and three virulence
loci (icmK, lspE, lssD), to genotype 110 L. pneumophila isolates from
various natural and artificial water sources in Guangdong province of China and
compared with the SBT. The isolates were assigned to 33 STs of the SBT and 91
new sequence types (nSTs) of the MLST. The indices of discrimination (IODs) of
SBT and MLST were 0.920 and 0.985, respectively. Maximum likelihood trees of the
concatenated SBT and MLST sequences both showed distinct phylogenetic
relationships between the isolates from the two environments. More intragenic
recombinations were detected in nSTs than in STs, and they were both more
abundant in natural water isolates. We found out the MLST had a high
discriminatory ability for the disease associated ST1 isolates: 22 ST1 isolates
were assigned to 19 nSTs. Furthermore, we assayed the discrimination of the MLST
for 29 reference strains (19 clinical and 10 environmental). The clinical
strains were assigned to eight STs and ten nSTs. The MLST could also subtype the
prevalent clinical ST36 or ST47 strains: eight ST36 strains were subtyped into
three nSTs and two ST47 strains were subtyped into two nSTs. We found different
distribution patterns of nSTs between the environmental and clinical ST36
isolates, and between the outbreak clinical ST36 isolates and the sporadic
clinical ST36 isolates. These results together revealed the MLST scheme could be
used as part of a typing scheme that increased discrimination when necessary.
Farhat M, Shaheed RA, Al-Ali HH,
Al-Ghamdi AS, Al-Hamaqi GM, Maan HS, Al-Mahfoodh ZA, Al-Seba HZ.
Department of Biochemistry, Imam
Abdulrahman Bin Faisal University, Dammam, Kindgdom of Saudi Arabia. mFarhat@uod.edu.sa
Saudi Med J 2018
Feb;39(2):137-141.
Abstract:
Objectives: To
investigate the presence of Legionella spp in cooling tower water. Legionella
proliferation in cooling tower water has serious public health implications as
it can be transmitted to humans via aerosols and cause Legionnaires' disease. Methods: Samples
of cooling tower water were collected from King Fahd Hospital of the University
(KFHU) (Imam Abdulrahman Bin Faisal University, 2015/2016). The water samples
were analyzed by a standard Legionella culture method, real-time
polymerase chain reaction (RT-PCR), and 16S rRNA next-generation sequencing. In
addition, the bacterial community composition was evaluated. Results: All
samples were negative by conventional Legionella culture. In contrast,
all water samples yielded positive results by real-time PCR (105 to 106 GU/L).
The results of 16S rRNA next generation sequencing showed high similarity and
reproducibility among the water samples. The majority of sequences were Alpha-,
Beta-, and Gamma-proteobacteria, and Legionella was the predominant genus.
The hydrogen-oxidizing gram-negative bacterium Hydrogenophaga was present at
high abundance, indicating high metabolic activity. Sphingopyxis, which is known
for its resistance to antimicrobials and as a pioneer in biofilm formation, was
also detected. Conclusion: Our
findings indicate that monitoring of Legionella in cooling tower water
would be enhanced by use of both conventional culturing and molecular methods.
Párraga-Niño N,
Quero S, Uria N, Castillo-Fernandez O, Jimenez-Ezenarro J, Muñoz FX, Sabrià
M, Garcia-Nuñez M.
Unitat de Malalties Infeccioses, Fundació
Institut d'Investigació Germans Trias I Pujol, Badalona, Spain. msabria.germanstrias@gencat.cat
Diagn
Microbiol Infect Dis 2018 Feb;90(2):85-89.
Abstract: Legionella pneumophila is
responsible for Legionnaires' disease (LD). Its detection in both environmental
and clinical samples is mainly performed by culture plate method which requires
up to 10days to obtain results. Nowadays, there are commercial antibodies
against this bacterium, but they have not been tested against all subgroups of L.
pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella
bacteria. Indeed, many of these antibodies became available when only 8
serogroups of L. pneumophila had been described. We tested 7 antibodies
and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L.
pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the
LP3IIG2 antibody detected almost all serogroups tested with lower rates of
cross-reactivity, resulting in a specific sensitive antibody for the detection
of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity
against respiratory non-Legionella isolates, thereby contraindicating its
clinical applicability.
Kober
C, Niessner R, Seidel M.
Institute of Hydrochemistry, Chair of Analytical
Chemistry and Water Chemistry, Technical University of Munich, München, Germany.
Michael.Seidel@ch.tum.de
Biosens
Bioelectron 2018 Feb;100:49-55.
Abstract: Increasing numbers of legionellosis
outbreaks within the last years have shown that Legionella are a growing
challenge for public health. Molecular biological detection methods capable of
rapidly identifying viable Legionella are important for the control of
engineered water systems. The current gold standard based on culture methods
takes up to 10 days to show positive results. For this reason, a flow-based
chemiluminescence (CL) DNA microarray was developed that is able to quantify
viable and non-viable Legionella spp. as well as Legionella
pneumophila in one hour. An isothermal heterogeneous asymmetric recombinase
polymerase amplification (haRPA) was carried out on flow-based CL DNA
microarrays. Detection limits of 87 genomic units (GU) µL-1 and
26GUµL-1 for Legionella spp. and Legionella
pneumophila, respectively, were achieved. In this work, it was shown for the
first time that the combination of a propidium monoazide (PMA) treatment with
haRPA, the so-called viability haRPA, is able to identify viable Legionella
on DNA microarrays. Different proportions of viable and non-viable Legionella,
shown with the example of L. pneumophila, ranging in a total
concentration between 101 to 105GUµL-1 were
analyzed on the microarray analysis platform MCR 3. Recovery values for viable Legionella
spp. were found between 81% and 133%. With the combination of these two
methods, there is a chance to replace culture-based methods in the future for
the monitoring of engineered water systems like condensation recooling plants.
Decker BK, Harris PL, Toy DL,
Muder RR, Sonel AF, Clancy CJ.
Infectious Diseases Section, Veterans
Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania. brooke.decker@va.gov
Infect Control Hosp Epidemiol 2018
Jan;39(1):108-110.
Abstract: Water cultures were significantly more
sensitive than concurrently collected swab cultures (n=2,147 each) in detecting Legionella
pneumophila within a Veterans Affairs healthcare system. Sensitivity for
water versus swab cultures was 90% versus 30% overall, 83% versus 48% during a
nosocomial Legionnaires' disease outbreak, and 93% versus 22% post outbreak.
Trnková K, Kotrbancová M, Špaleková
M, Fulová M, Boledovičová J, Vesteg M.
Department of the Environment, Faculty of Natural
Sciences, Matej Bel University, Tajovského 55, 974 01 Banská Bystrica,
Slovakia. katarina.trnkova@umb.sk
Exp Parasitol 2018
Jan;184:97-102.
Abstract: Legionellae, i.e. Legionella
pneumophila, are human bacterial hydrophilic facultative pathogens causing
pneumonia (Legionnaires' disease). Free-living amoebae (FLA) can serve as
natural hosts and thus as reservoirs of many amoebae-resistant bacteria. An
encysted amoeba can contribute to the resistance of intracellular L.
pneumophila to various chemical and physical treatments. Humans can be
infected by droplets containing bacteria from an environmental source or
human-made devices such as shower heads, bathtubs, air-conditioning units or
whirlpools. In this study, we were investigating the presence of FLA and L.
pneumophila in plumbing systems of healthcare facilities in Bratislava (Slovakia)
by standard diagnostic methods, while the presence of L. pneumophila was
verified also by MALDI-TOF MS (matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry) analysis. The results showed the occurrence of
L. pneumophila and FLA in 62.26% and 66.4% of samples taken from four
pediatric clinics, respectively. Both standard methods and MALDI-TOF MS showed
comparable results and they can be successfully applied for the identification
of L. pneumophila strains in environmental samples. Our approach could be
useful for further monitoring, prevention and decreasing risk of Legionella infection
also in other hospitals.
Xi
D, Dou Y, Ren W, Yang S, Feng L, Cao B, Wang L.
The Key Laboratory of Molecular Microbiology and
Technology of the Ministry of Education, Nankai University, Tianjin, China. wanglei@nankai.edu.cn
Antonie Van
Leeuwenhoek 2017 Dec;110(12):1515-1525.
Abstract: Among the 50 species and 70 serogroups
of Legionella identified, Legionella pneumophila, comprising three
subsp. (subsp. pneumophila, subsp. fraseri, and subsp. pasculleii),
is recognized as the major cause of epidemic legionellosis. Rapid and reliable
assays to identify pathogenic Legionella spp., and the three L.
pneumophila subsp. in particular, are in great demand. In this study, we
analyzed the gyrB genes of eleven Legionella spp. and subsp., comprising L.
anisa, L. bozemanii, L. dumoffii, L. feeleii, L.
gormanii, L. longbeachae, L. micdadei, L. waltersii, L.
pneumophila subsp. pneumophila, L. pneumophila subsp. fraseri,
and L. pneumophila subsp. pasculleii. We developed a rapid
oligonucleotide microarray detection technique to identify accurately these
common pathogenic Legionella spp. and L. pneumophila subsp. To
detect multiple Legionella species with high specificity, 31 reproducible
probes were designed in the array. Sixty-one strains were analyzed in total,
including 37 target pathogens and 24 non-target bacterial species used to
validate the microarray. The sensitivity of the detection was 1.0 ng using
genomic DNA of three Legionella spp., L. anisa, L. dumoffii,
and L. waltersii, or 13 CFU/100 mL using the cultured L. pneumophila
subsp. pneumophila. Eight isolated strains were tested using the
microarray with 100% accuracy. The data indicated that the technique is an
efficient method to diagnose and detect Legionella spp. and subsp. in
basic microbiology, clinical diagnosis, epidemiological surveillance, and food
safety applications. In addition, a phylogenetic study based on the gyrB gene
revealed the genetic relationship among the different Legionella spp. and
subsp.
Ranjbar
R, Behzadi P, Najafi A, Roudi R.
Department of Microbiology, College of Basic Sciences,
Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran. behzadipayam@yahoo.com
Open
Microbiol J 2017 Nov;11:330-338.
Abstract: Background: A
rapid, accurate, flexible and reliable diagnostic method may significantly
decrease the costs of diagnosis and treatment. Designing an appropriate
microarray chip reduces noises and probable biases in the final result. Objective: The
aim of this study was to design and construct a DNA Microarray Chip for a rapid
detection and identification of 10 important bacterial agents. Method: In
the present survey, 10 unique genomic regions relating to 10 pathogenic
bacterial agents including Escherichia coli (E.coli), Shigella boydii,
Sh. dysenteriae, Sh. flexneri, Sh. sonnei, Salmonella typhi, S. typhimurium,
Brucella sp., Legionella pneumophila, and Vibrio cholera were
selected for designing specific long oligo microarray probes. For this reason,
the in-silico operations including utilization of the NCBI RefSeq database,
Servers of PanSeq and Gview, AlleleID 7.7 and Oligo Analyzer 3.1 was done. On
the other hand, the in-vitro part of the study comprised stages
of robotic microarray chip probe spotting, bacterial DNAs extraction and DNA
labeling, hybridization and microarray chip scanning. In wet lab section,
different tools and apparatus such as Nexterion® Slide E, Qarraymini spotter,
NimbleGen kit, TrayMixTM S4, and Innoscan 710 were used. Results: A
DNA microarray chip including 10 long oligo microarray probes was designed and
constructed for detection and identification of 10 pathogenic bacteria. Conclusion: The
DNA microarray chip was capable to identify all 10 bacterial agents tested
simultaneously. The presence of a professional bioinformatician as a probe
designer is needed to design appropriate multifunctional microarray probes to
increase the accuracy of the outcomes.
Lizana
X, López A, Benito S, Agustí G, Ríos M, Piqué
N, Marqués AM, Codony F.
GenIUL,
Carrer de la Ciutat d'Asunción, Barcelona, Spain. fcodony@geniul.com
Int J Hyg
Environ Health 2017 Nov;220(8):1318-1324.
Abstract: Background: Viability
quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach
for only detecting live microorganisms by DNA amplification-based methods This
approach is based on the use of a reagent that irreversibly fixes dead cell’s
DNA. In this study, we evaluate the utility of v-qPCR versus culture method for
Legionellosis risk management. Methods: The
present study was performed using 116 real samples. Water samples were
simultaneously analysed by culture, v-qPCR and qPCR methods. Results were
compared by means of a non-parametric test. Results: In
11.6% of samples using both methods (culture method and v-qPCR) results were
positive, in 50.0% of samples both methods gave rise to negative results. As
expected, equivalence between methods was not observed in all cases, as in 32.1%
of samples positive results were obtained by v-qPCR and all of them gave rise to
negative results by culture. Only in 6.3% of samples, with very low Legionella
levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth
of other bacteria did not allow performing the culture. When comparing both
methods, significant differences between culture and v-qPCR were in the samples
belonging to the cooling towers-evaporative condensers group. The v-qPCR method
detected greater presence and obtained higher concentrations of Legionella
spp. (p<0.001). Otherwise, no significant differences between methods were
found in the rest of the groups. Conclusions: The v-qPCR method can be
used as a quick tool to evaluate Legionellosis risk, especially in cooling
towers-evaporative condensers, where this technique can detect higher levels
than culture. The combined interpretation of PCR results along with the ratio of
live cells is proposed as a tool for understanding the sample context and
estimating the Legionellosis risk potential according to 4 levels of hierarchy.
Ranc AG, Carpentier M, Beraud
L, Descours G, Ginevra C, Maisonneuve E, Verdon J, Berjeaud JM, Lina G, Jarraud
S.
Hospices Civils de Lyon, Centre National
de Référence des Légionelles, France.
Diagn Microbiol Infect Dis 2017 Oct;89 (2):89-91.
Abstract: Three urinary antigen tests were
compared using purified Legionella pneumophila (Lp) LPS. For Lp
serogroup1, Sofia®FIA and Binax®EIA limits of detection (LOD) were similar;
that of BinaxNOW® lower. For all tests the LOD was higher with LPS from
non-Pontiac compared to Pontiac-strains. The LOD was variable for other Lp
serogroups.
Athlin S, Iversen A, Özenci V.
Department of Infectious Diseases,
Faculty of Medicin and Health, Örebro University, Örebro, Sweden. simon.athlin@regionorebrolan.se
Eur J Clin
Microbiol Infect Dis 2017 Oct;36(10):1933-1938.
Abstract: The use of urinary antigen tests (UATs)
may provide early etiology in pneumonia and facilitates rapid and directed
antibiotic treatment. In this study, we evaluated the novel lateral flow
ImmuView Streptococcus pneumoniae and Legionella pneumophila UAT,
which detects pneumococcal and L. pneumophila serogroup 1 antigens in a
combined test. We compared the ImmuView UAT with the BinaxNOW S. pneumoniae
UAT and the BinaxNOW L. pneumophila UAT in 147 patients with pneumococcal
bacteremia (n=48), non-pneumococcal non-Legionella bacteremia (n=93) and Legionella
infections in the lower airways (L. pneumophila, n=5; L. bozemanii,
n=1). In three cases, the ImmuView test was invalid before and after boiling
while the BinaxNOW tests were valid in all cases. In 144 cases, the three UATs
demonstrated a very good inter-assay agreement for detection of pneumococcal
antigen (κ=0.86) and L. pneumophila antigen (κ=1.00). The ImmuView and BinaxNOW S. pneumoniae
tests had similar sensitivities (62% vs 60%; p=ns) in 48 cases with pneumococcal
bacteremia and both tests had specificities of 97% in 96 cases with
non-pneumococcal infections. Furthermore, the ImmuView and BinaxNOW L.
pneumophila tests were positive for Legionella antigen in five
patients with confirmed L. pneumophila serogroup 1 infections, and
negative in all non-L. pneumophila cases. The ImmuView and BinaxNOW tests
performed similarly when evaluated on urine samples from bacteremic and
non-bacteremic patients with identified etiology.
Veenendaal
HR, Brouwer-Hanzens AJ, van der Kooij D.
KWR Watercycle Research Institute,
Nieuwegein, The Netherlands. dick.van.der.kooij@kwrwater.nl
Water Res 2017 Oct;123:439-447.
Abstract: Worldwide, over 90% of the notified
cases of Legionnaires' disease are caused by Legionella pneumophila.
However, the standard culture medium for the detection of Legionella in
environmental water samples, Buffered Charcoal Yeast Extract (BCYE) agar of pH
6.9±0.4 with or without antimicrobial agents incubated at 36±1°C, supports
the growth of a large diversity of Legionella species. BCYE agar of
elevated pH or/and incubation at elevated temperature gave strongly reduced
recoveries of most of 26 L. non-pneumophila spp. tested, but not
of L. pneumophila. BCYE agar of pH 7.3±0.1, incubated at 40±0.5°C (BCYE
pH 7.3/40°C) was tested for selective enumeration of L. pneumophila. Of
the L. non-pneumophila spp. tested, only L. adelaidensis
and L. londiniensis multiplied under these conditions. The colony counts
on BCYE pH 7.3/40°C of a L. pneumophila serogroup 1 strain cultured in
tap water did not differ significantly from those on BCYE pH 6.9/36°C when
directly plated and after membrane filtration and showed repeatability's of
13-14%. By using membrane filtration L. pneumophila was detected in 58
(54%) of 107 Legionella-positive water samples from premise plumbing
systems under one or both of these culture conditions. The L. pneumophila
colony counts (log-transformed) on BCYE pH 7.3/40°C were strongly related (r2=0.87)
to those on BCYE pH 6.9/36°C but differed significantly (p<0.05) by a mean
of -0.12±0.30 logs. L. non-pneumophila spp. were detected only on
BCYE pH 6.9/36°C in 49 (46%) of the samples. Hence, BCYE pH 7.3/40°C can
facilitate the enumeration of L. pneumophila and their isolation from
premise plumbing systems with culturable L. non-pneumophila spp.,
some of which, e.g. L. anisa, can be present in high numbers.
Marinelli L, Cottarelli A, Solimini
AG, Del Cimmuto A, De Giusti M.
Department of Public Health and Infectious Diseases,
Sapienza University of Rome, Italy.
lucia.marinelli@uniroma1.it
Ann Ig 2017
Sep-Oct;29(5):431-439.
Abstract: Background: In
this study we estimated the presence of Legionella species, viable but
non-culturable (VBNC), in hospital water networks. We also evaluated the time
and load of Legionella appearance in samples found negative using the
standard culture method. Methods: A
total of 42 samples was obtained from the tap water of five hospital buildings.
The samples were tested for Legionella by the standard culture method and
were monitored for up to 12 months for the appearance of VBNC Legionella. Results: All
the 42 samples were negative at the time of collection. Seven of the 42 samples
(17.0%) became positive for Legionella at different times of monitoring.
The time to the appearance of VBNC Legionella was extremely variable,
from 15 days to 9 months from sampling. The most frequent Legionella species
observed were Legionella spp and L. anisa and only in one sample L.
pneumophila srg.1. Conclusions: Our
study confirms the presence of VBNC Legionella in samples resulting
negative using the standard culture method and highlights the different time to
its appearance that can occur several months after sampling. The results are
important for risk assessment and risk management of engineered water systems.
Samhan
FA, Stedtfeld TM, Waseem H, Williams MR,
Stedtfeld RD, Hashsham SA.
Department of Civil and Environmental Engineering,
Michigan State University, East Lansing, MI, USA. hashsham@egr.msu.edu
Water Res 2017
Sep;121:162-170.
Abstract: Guidelines and regulations to control Legionella
pneumophila in cooling water systems of large buildings are evolving due to
the increasing number of outbreaks. Rapid, on-site, simple, and sensitive
quantification methods that are also able to assess viability may be extremely
useful in monitoring and control. Culture-based methods for measuring L.
pneumophila may take 4-10 days and qPCR-based methods are also slow,
requiring at least a day from sample to result, albeit mainly due to the need
for sample transport to a centralized laboratory. This study reports a rapid
isothermal amplification method for L. pneumophila concentration and
detection with live/dead differentiation under field conditions. Using an
on-filter direct amplification (i.e., amplification of cells without DNA
extraction and purification) approach with propidium monoazide (PMA), and a real
time isothermal amplification platform (Gene-Z), L. pneumophila could be
detected in 1-2 h at ∼1 cfu/100 ml of tap
water. Signature sequences from 16S rRNA and cadA genes were used as genetic
markers for L. pneumophila and loop-mediated isothermal amplification (LAMP)
primers were designed using Primer Explorer V4. Result were also compared with
direct amplification of cells spiked into distilled, tap, and cooling water
samples as well as extracted DNA by qPCR. This method may be useful to managers
of cooling water systems in large buildings for rapid detection of L.
pneumophila. The overall approach of on-site sample concentration, on-filter
amplification, and live/dead differentiation may be extended to other organisms
where analytical sensitivity and speed are equally important.
Valavane A,
Chaudhry R, Malhotra P.
Department of Microbiology, All India Institute of
Medical Sciences, New Delhi, India. drramach@gmail.com
Indian J Med
Res 2017 Sep;146(3):392-400.
Abstract: Background &
objectives: Genomic
constitution of the bacterium Legionella pneumophila plays an important
role in providing them a pathogenic potential. Here, we report the
standardization and application of multiplex polymerase chain reaction (PCR) for
the detection of molecular markers of pathogenic potential in L. pneumophila
in hospital environment. Methods: Culture
of the standard strains of L. pneumophila was performed in buffered
charcoal-yeast extract agar with L-cysteine at pH 6.9. Primers were designed for
multiplex PCR, and standardization for the detection of five markers annotated
to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis
(19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling
factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water
samples and 200 swabs were collected from the hospital environment. The
bacterium was isolated from the hospital environment by culture and confirmed by
16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L.
pneumophila isolates were studied using the standardized multiplex PCR. Results: The
PCR was sensitive to detect 0.1 ng/μl
DNA and specific for the two standard strains used in the study. Of the 45
hospital isolates tested, 11 isolates had four markers, 12 isolates had three
markers, 10 isolates had two markers, nine isolates had one marker and three
isolates had none of the markers. None of the isolates had all the five markers.
Interpretation & conclusions: The
findings of this study showed the presence of gene markers of pathogenic
potential of the bacterium L. pneumophila. However, the genomic
constitution of the environmental isolates should be correlated with clinical
isolates to prove their pathogenic potential. Rapid diagnostic methods such as
multiplex PCR reported here, for elucidating gene markers, could help in future
epidemiological studies of bacterium L. pneumophila.
Pierre
DM, Baron J, Yu VL, Stout JE.
Department of Medicine, School of
Medicine, University of Pittsburgh, Pittsburgh, PA, USA. victorlyu@gmail.com
Ann Clin
Microbiol Antimicrob 2017 Aug;16(1):59.
Abstract: Legionnaires' disease is commonly
diagnosed clinically using a urinary antigen test. The urinary antigen test is
highly accurate for L. pneumophila serogroup 1, however other diagnostic
tests should also be utilized in conjunction with the urinary antigen as many
other Legionella species and serogroups are pathogenic. Culturing of
patient specimens remains the gold standard for diagnosis of Legionnaires'
disease. Selective media, BYCE with the addition of antibiotics, allows for a
high sensitivity and specificity. Culturing can identify all species and
serogroups of Legionella. A major benefit of culturing is that it
provides the recovery of a patient isolate, which can be used to find an
environmental match. Other diagnostic tests, including DFA and molecular tests
such as PCR and LAMP, are useful tests to supplement culturing. Molecular tests
provide much more rapid results in comparison to culture; however, these tests
should not be a primary diagnostic tool given their lower sensitivity and
specificity in comparison to culturing. It is recommended that all laboratories
develop the ability to culture patient specimens in-house with the selective
media.
Hennebique A,
Bidart M, Jarraud S, Beraud L, Schwebel C, Maurin M, Boisset S.
Institut de Biologie et de Pathologie,
Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France. ahennebique@chu-grenoble.fr e sboisset@chu-grenoble.fr
Antimicrob
Agents Chemother 2017 Aug;61(9):e00628-17.
Abstract: The emergence of fluoroquinolone (FQ)-resistant
mutants of Legionella pneumophila in infected humans was
previously reported using a next-generation DNA sequencing (NGS) approach. This
finding could explain part of the therapeutic failures observed in legionellosis
patients treated with these antibiotics. The aim of this study was to develop
digital PCR (dPCR) assays allowing rapid and accurate detection and
quantification of these resistant mutants in respiratory samples, especially
when the proportion of mutants in a wild-type background is low. We designed
three dPCRgyrA assays to detect and differentiate the wild-type and one of the
three gyrA mutations previously described as associated with FQ
resistance in L. pneumophila: at positions 248C→T (T83I), 259G→A (D87N), and 259G→C (D87H). To assess the performance of these assays,
mixtures of FQ-resistant and -susceptible strains of L. pneumophila were
analyzed, and the results were compared with those obtained with Sanger DNA
sequencing and real-time quantitative PCR (qPCR) technologies. The dPCRgyrA
assays were able to detect mutated gyrA sequences in the
presence of wild-type sequences at up to 1:1,000 resistant/susceptible allele
ratios. By comparison, Sanger DNA sequencing and qPCR were less sensitive,
allowing the detection of gyrA mutants at up to 1:1 and 1:10
ratios, respectively. When testing 38 respiratory samples from 23 legionellosis
patients (69.6% treated with an FQ), dPCRgyrA detected small amounts of gyrA mutants
in four (10.5%) samples from three (13.0%) patients. These results demonstrate
that dPCR is a highly sensitive alternative to quantify FQ resistance in L.
pneumophila, and it could be used in clinical practice to detect patients
that could be at higher risk of therapeutic failure.
Gong
X, Li J, Zhang Y, Hou S, Qu P, Yang Z, Chen S.
Guangzhou Center for Disease Control and
Prevention, Guangzhou, China. shouyi_chen@163.com
J Microbiol
Methods 2017 Aug;139:1-7.
Abstract: Legionella spp. are important
waterborne pathogens. Molecular typing has become an important method for
outbreaks investigations and source tracking of Legionnaires’ disease. In a
survey program conducted by the Guangzhou Center for Disease Control and
Prevention, multiple serotypes Legionella pneumophila (L. pneumophila)
were isolated from waters in air-conditioning cooling towers in urban Guangzhou
region, China between 2008 and 2011. Three genotyping methods, mip (macrophage
infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent
amplified fragment length polymorphism analysis) were used to type these
waterborne L. pneumophila isolates. The three methods were capable of
typing all the 134 isolates and a reference strain of L. pneumophila
(ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the
mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134
isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and
FAFLP methods respectively. The mip genotyping and SBT typing are more feasible
for inter-laboratory results sharing and comparison of different types of L.
pneumophila. The SBT and FAFLP typing methods were rapid with higher
discriminatory abilities. Combinations of two or more of the typing methods
enables more accurate typing of Legionella isolates for outbreak
investigations and source tracking of Legionnaires’ disease.
Osborne AJ,
Jose BR, Perry J, Smeele Z, Aitken J, Gardner PP, Slow S.
School of Biological Sciences, University
of Canterbury, Christchurch, New Zealand. amy.osborne@otago.ac.nz
Genome
Announc 2017 Jun;5(22):e00436-17.
Abstract:
Legionella is a highly diverse genus of intracellular bacterial
pathogens that cause Legionnaire's disease (LD), an often-severe form of
pneumonia. Two L. micdadei sp. clinical isolates, obtained from
patients hospitalized with LD from geographically distinct areas, were sequenced
using PacBio SMRT cell technology, identifying incomplete phage regions, which
may impact virulence.
Slow S, Anderson T, Miller J,
Singh S, Murdoch D, Biggs PJ.
Department of Pathology, University of
Otago, Christchurch, New Zealand. sandy.slow@otago.ac.nz
Genome
Announc 2017 Jun;5(24):e00564-17.
Abstract: Legionella longbeachae serogroup
1, predominantly found in soil and composted plant material, causes the majority
of cases of Legionnaires' disease (LD) in New Zealand. Here, we report the
complete genome sequence of an L. longbeachae serogroup 1 (sg1)
isolate derived from a patient hospitalized with LD in Christchurch, New Zealand.
Montagna MT, De Giglio O, Cristina ML,
Napoli C, Pacifico C, Agodi A, Baldovin T, Casini B, Coniglio MA, D'Errico MM,
Delia SA, Deriu MG, Guida M, Laganà P, Liguori G, Moro M, Mura I, Pennino F,
Privitera G, Romano Spica V, Sembeni S, Spagnolo AM, Tardivo S, Torre I,
Valeriani F, Albertini R, Pasquarella C.
Department of Biomedical Science and
Human Oncology, University of Bari "Aldo Moro", Bari, Italy. mariateresa.montagna@uniba.it
Int J Environ
Res Public Health 2017 Jun;14(7):670.
Abstract: Healthcare facilities (HF) represent an
at-risk environment for legionellosis transmission occurring after inhalation of
contaminated aerosols. In general, the control of water is preferred to that of
air because, to date, there are no standardized sampling protocols. Legionella air
contamination was investigated in the bathrooms of 11 HF by active sampling (Surface
Air System and Coriolis®μ)
and passive sampling using settling plates. During the 8-hour sampling, hot tap
water was sampled three times. All air samples were evaluated using
culture-based methods, whereas liquid samples collected using the Coriolis®μ were also analyzed by real-time PCR. Legionella presence
in the air and water was then compared by sequence-based typing (SBT) methods.
Air contamination was found in four HF (36.4%) by at least one of the culturable
methods. The culturable investigation by Coriolis®μ did not yield Legionella in any
enrolled HF. However, molecular investigation using Coriolis®μ resulted in eight HF testing positive for Legionella in
the air. Comparison of Legionella air and water contamination
indicated that Legionella water concentration could be
predictive of its presence in the air. Furthermore, a molecular study of 12 L.
pneumophila strains confirmed a match between the Legionella strains
from air and water samples by SBT for three out of four HF that tested positive
for Legionella by at least one of the culturable methods.
Overall, our study shows that Legionella air detection cannot
replace water sampling because the absence of microorganisms from the air does
not necessarily represent their absence from water; nevertheless, air sampling
may provide useful information for risk assessment. The liquid impingement
technique appears to have the greatest capacity for collecting airborne Legionella if
combined with molecular investigations.
Collins S,
Stevenson D, Walker J, Bennett A.
Biosafety Air and Water Microbiology Group, Public
Health England, Porton Down, Salisbury, UK. samuel.collins@phe.gov.uk
J Appl
Microbiol 2017 Jun;122(6):1692-1703.
Abstract: Aims: To
evaluate the usefulness of Legionella qPCR alongside traditional culture for
enumeration of Legionella from water samples as part of both routine and
public health investigation testing. Methods and
results: Routine
water samples (n=2002) and samples from public health investigations (n=215)
were analysed by culture and qPCR for Legionella spp., Legionella
pneumophila and L. pneumophila sg 1. A negative qPCR result was
highly predictive of a negative culture result for all water systems (negative
predictive values, NPV from 97.4 to 100%). Positive predictive values (PPV) were
lower (0-50%). Results for qPCR were generally larger than culture with average
log10 differences of 1.1 for Legionella spp. and 1.2 for L.
pneumophila. Alert and action levels of 1000 and 10 000 GU per litre,
respectively, are proposed for Legionella qPCR for hot and cold water systems (HCWS).
The use of qPCR significantly reduced the time to results for public health
investigations by rapidly identifying potential sources and ruling out others,
thus enabling a more rapid and efficient response. Conclusions: The high NPV of qPCR
supports its use to rapidly screen out negative samples without culture. Alert
and action levels for Legionella qPCR for HCWS are proposed. Quantitative PCR
will be a valuable tool for both routine and public health testing. Significance and impact of the study: This
study generated comparative data of >2000 water samples by qPCR and culture.
Action and alert levels have been recommended that could enable duty holders to
interpret qPCR results to facilitate timely Legionella control and public
health protection.
Badoux P, Euser SM, Bruin JP,
Mulder PPG, Yzerman EPF.
Regional Public Health Laboratory
Kennemerland, Boerhaavelaan, Haarlem, the Netherlands. p.badoux@streeklabhaarlem.nl
J Clin
Microbiol 2017 Jun;55(6):1733-1737.
Abstract: In this study, we compared the bioNexia
test (bioMérieux, Marcy-l'Étoile, France), a new immunochromatographic assay
for the detection of Legionella pneumophila serogroup 1 in
urine, with the BinaxNOW urinary antigen test (Alere, Waltham, Massachusetts,
USA). After 15 min of incubation (in accordance with the manufacturers'
instructions), the sensitivities and specificities were, respectively, 76.5% and
97.2% for the bioNexia test and 87.1% and 100% for the BinaxNOW test. After a
prolonged incubation time of 60 min, the sensitivities and specificities
increased to, respectively, 89.4% and 97.2% for the bioNexia test and 91.8% and
100% for the BinaxNOW test. When the tests were read after 15 min, the
concentration of discrepant urine samples increased the sensitivities to 94.1%
for both tests. In conclusion, we found that although the bioNexia test showed
lower sensitivity for the detection of L. pneumophila antigen
in nonconcentrated urine compared to the BinaxNOW test, a prolonged incubation
time as well as the use of concentrated samples showed comparable sensitivities
for both tests.
Yamaguchi N,
Tokunaga Y, Goto S, Fujii Y, Banno F, Edagawa A.
Osaka Institute of Public Health, Osaka, Japan. nyyamaguchi@iph.osaka.jp
Sci Rep 2017
Jun;7(1):3092.
Abstract: Legionnaires' disease, predominantly
caused by the bacterium Legionella pneumophila, has increased in
prevalence worldwide. The most common mode of transmission of Legionella
is inhalation of contaminated aerosols, such as those generated by cooling
towers. Simple, rapid and accurate methods to enumerate L. pneumophila
are required to prevent the spread of this organism. Here, we applied a
microfluidic device for on-chip fluorescent staining and semi-automated counting
of L. pneumophila in cooling tower water. We also constructed a portable
system for rapid on-site monitoring and used it to enumerate target bacterial
cells rapidly flowing in the microchannel. A fluorescently labelled polyclonal
antibody was used for the selective detection of L. pneumophila serogroup
1 in the samples. The counts of L. pneumophila in cooling tower water
obtained using the system and fluorescence microscopy were similar. The
detection limit of the system was 104 cells/ml, but lower
numbers of L. pneumophila cells (101 to 103 cells/ml)
could be detected following concentration of 0.5-3 L of the water sample by
filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip
staining and counting), and portable for on-site measurement, and it may
therefore be effective in the initial screening of Legionella
contamination in freshwater.
Gordon
M, Yakunin E, Valinsky L, Chalifa-Caspi V, Moran-Gilad
J; ESCMID Study Group for Legionella Infections.
Ben-Gurion University of the Negev, Beer-Sheva, Israel.
giladko@post.bgu.ac.il
Clin
Microbiol Infect 2017 May;23(5):306-310.
Abstract: Objectives: Whole
genome sequencing (WGS) has revolutionized the subtyping of Legionella
pneumophila but calling the traditional sequence-based type from genomic
data is hampered by multiple copies of the mompS locus. We propose a novel
bioinformatics solution for rectifying that limitation, ensuring the feasibility
of WGS for cluster investigation. Methods: We
designed a novel approach based on the alignment of raw reads with a reference
sequence. With WGS, reads originating from either of the two mompS copies cannot
be differentiated. Therefore, when non-identical copies were present, we applied
a read-filtering strategy based on read alignment to a reference sequence via
unique 'anchors'. If minimal read coverage was achieved after filtration
(≥3X), a consensus sequence was built based on mapped reads followed by
calling the sequence-based typing allele. The entire procedure was implemented
using a Perl script. Results: The
method was validated using a diverse sample of 265 L. pneumophila genomes,
consisting of 59 different sequence types (STs) and 23 mompS variants; 57 of the
265 (22%) had non-identical mompS copies. In 237 of the 265 samples (89.4%),
mompS calling was successful and no erroneous calling occurred. A 98.1% success
was recorded among 109 samples meeting quality requirements. The method was
superior to alternative approaches. Conclusions: As
WGS becomes more accessible, technical difficulties in routine clinical and
surveillance work will arise. The case of mompS in L. pneumophila serves
as an example for such limitations that necessitate the development of novel
computational solutions that meet end-user demands.
Miyata J, Huh JY, Ito Y, Kobuchi T, Kusukawa K, Hayashi H.
Division
of Family Medicine University of Fukui Hospital Fukui Japan. j-miyata@umin.ac.jp
J Gen Fam Med
2017 Apr;18(3):139-143.
Abstract: It is critical to diagnose and treat Legionella
pneumonia (LP) immediately after infection because of the associated high
mortality. The urine antigen test (UAT) is often used for the diagnosis of LP;
however, it cannot detect the serogroups of all Legionella species. A
detained medical history and several clinical findings such as liver enzyme
elevation and hyponatremia are useful in diagnosis. Some specific types of Legionella are
found in compost. Herein, we report a case of LP in which the patient's medical
history and several clinical findings were useful for diagnosis.
Cristovam E, Almeida D, Caldeira D,
Ferreira JJ, Marques T.
Laboratory of Microbiology and Molecular Biology,
Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal. elisabete.cristovam@gmail.com
J Med
Microbiol 2017 Apr;66(4):485-489.
Abstract: Purpose: Rapid
and effective diagnosis of Legionnaires' disease (LD) cases is extremely
important so that timely and appropriate therapy can be provided, thereby
lowering the morbidity and mortality rates and reducing the health and economic
costs associated with this disease. Methodology: Diagnosis
is established solely by microbiological tests. There are several methods
available, each with different performance, sensitivity and specificity
characteristics, and further understanding is required. Our objective was to
assess the accuracy of urinary antigen detection, direct fluorescent antibody (DFA)
staining, serological testing and the polymerase chain reaction (PCR) method
versus culture analysis (the reference standard) in patients suspected of being
infected with Legionella or patients with laboratory-confirmed LD. We
performed a MEDLINE search in November 2014. Two authors independently assessed
the trials and extracted data. Pooled analysis was performed through Meta-DiSc
version 1.4. Result: The
inclusion criteria were met by 11 studies. All the studies evaluated PCR and DFA
tests to detect Legionella in clinical specimens, comparing them to
culture techniques, and were included in the meta-analysis. The pooled
sensitivity and specificity for PCR were 83 % [95% confidence interval (CI):
79-87%] and 90% (95% CI: 88-92%), respectively. DFA was evaluated in one study
and the sensitivity and specificity of this test were 67% (95% CI: 30-93%) and
100% (95% CI: 91-100%), respectively. PCR had high sensitivity and specificity
for early diagnosis of LD. Conclusion: Culture
analysis is deemed necessary for epidemiological studies, molecular strain
typing and antibiotic sensibility evaluations; however, the performance of PCR
in recent studies calls for additional, well-designed studies in order to
achieve the best standard test, which will enable optimization of the Legionella
infection diagnostic.
Bonetta
S, Pignata C, Bonetta
S, Meucci L, Giacosa D, Marino E, Gilli G,
Carraro E.
Department of Public Health and
Pediatrics, University of Torino, Torino, Italy. sara.bonetta@unito.it
Int J Environ
Res Public Health 2017 Apr;14(5): 467.
Abstract: Legionella pneumophila is a
ubiquitous microorganism widely distributed in aquatic environments and can
cause Legionellosis in humans. A promising approach to detect viable cells in
water samples involves the use of quantitative polymerase chain reaction (qPCR)
in combination with photoactivatable DNA intercalator propidium monoazide (PMA).
However, the PMA efficiency could be different depending on the experimental
conditions used. The aim of this study was to compare two PMA exposure protocols:
(A) directly on the membrane filter or (B) in liquid after filter washing. The
overall PMA-induced qPCR means reductions in heat-killed L. pneumophila cells
were 2.42 and 1.91 log units for exposure protocols A and B, respectively. A
comparison between the results obtained reveals that filter exposure allows a
higher PMA-qPCR signal reduction to be reached, mainly at low concentrations (p<0.05).
This confirms the potential use of this method to quantify L.
pneumophila in water with low contamination.
Sartory DP,
Spies K, Lange B, Schneider S, Langer B.
SWM Consulting, Little Ness, Shrewsbury, UK. david.sartory@tesco.net
Lett Appl
Microbiol 2017 Apr;64(4):271-275.
Abstract: This study compared the performance of a
novel MPN method (Legiolert/Quanti-Tray) with the ISO 11731-2 membrane
filtration method for the enumeration of Legionella pneumophila from 100
ml potable water and related samples. Data from a multi-laboratory study
analysed according to ISO 17994 showed that Legiolert™/Quanti-Tray® yielded
on average higher counts of L. pneumophila. The Legiolert medium had a
high specificity of 96·4%. The new method represents a significant improvement
in the enumeration of L. pneumophila from drinking water-related samples.
Significance and impact of the study: Legionella
pneumophila is an opportunistic pathogen of major concern. The current large
volume quantitative method employs membrane filtration (MF) and selective
culture on GVPC agar followed by confirmation of isolates by serology (ISO
11731-2). We present here the results of a multi-laboratory evaluation of a most
probable number (MPN) in-situ confirmed method (Legiolert™/Quanti-Tray®). The
results indicate that Legiolert/Quanti-Tray yielded on average higher counts of L.
pneumophila than ISO 11731-2. This development significantly improves and
simplifies the enumeration of L. pneumophila from potable water samples.
Pereira RP,
Peplies J, Brettar I, Höfle MG.
Department of Vaccinology and Applied Microbiology, RG
Microbial Diagnostics, Helmholtz Centre for Infection Research (HZI),
Braunschweig, Germany. manfred.hoefle@helmholtz-hzi.de
BMC Microbiol
2017 Mar;17(1):79.
Abstract:
Background: Next
Generation Sequencing (NGS) has revolutionized the analysis of natural and
man-made microbial communities by using universal primers for bacteria in a PCR
based approach targeting the 16S rRNA gene. In our study we narrowed primer
specificity to a single, monophyletic genus because for many questions in
microbiology only a specific part of the whole microbiome is of interest. We
have chosen the genus Legionella, comprising more than 20 pathogenic
species, due to its high relevance for water-based respiratory infections. Methods: A
new NGS-based approach was designed by sequencing 16S rRNA gene amplicons
specific for the genus Legionella using the Illumina MiSeq technology.
This approach was validated and applied to a set of representative freshwater
samples. Results: Our
results revealed that the generated libraries presented a low average raw error
rate per base (<0.5%); and substantiated the use of high-fidelity enzymes,
such as KAPA HiFi, for increased sequence accuracy and quality. The approach
also showed high in situ specificity (>95%) and very good repeatability. Only
in samples in which the gamma bacterial clade SAR86 was present more than 1%
non-Legionella sequences were observed. Next-generation sequencing read
counts did not reveal considerable amplification/sequencing biases and showed a
sensitive as well as precise quantification of L. pneumophila along a
dilution range using a spiked-in, certified genome standard. The genome standard
and a mock community consisting of six different Legionella species
demonstrated that the developed NGS approach was quantitative and specific at
the level of individual species, including L. pneumophila. The
sensitivity of our genus-specific approach was at least one order of magnitude
higher compared to the universal NGS approach. Comparison of quantification by
real-time PCR showed consistency with the NGS data. Overall, our NGS approach
can determine the quantitative abundances of Legionella species, i.e. the
complete Legionella microbiome, without the need for species-specific
primers. Conclusions: The
developed NGS approach provides a new molecular surveillance tool to monitor all
Legionella species in qualitative and quantitative terms if a spiked-in
genome standard is used to calibrate the method. Overall, the genus specific NGS
approach opens up a new avenue to massive parallel diagnostics in a
quantitative, specific and sensitive way.
Dunne WM Jr, Picot N, van Belkum A.
Scientific Office, BioMérieux, 3, Route de Port
Michaud, La Balme Les Grottes 38390, France. alex.vanbelkum@biomerieux.com
Infect Dis
Clin North Am 2017 Mar;31(1):167-178.
Abstract: Legionella pneumophila is one of
the more recently discovered bacterial pathogens of humans. The last 2 decades
have seen tremendous progress in the evolution of diagnostic tests, for
detection and characterization of this pathogen and for defining the host
response to infection. This has generated several diagnostic tools that span the
range from simple immunologic assays to modern genome sequencing. This review
describes the state of affairs of this continuously evolving field regarding the
diagnosis of Legionnaire's disease and covers detection, assessment of
antibiotic susceptibility, and epidemiologic characterization of isolates of L.
pneumophila and other pathogenic species within the genus.
Yamakuchi H,
Hamada Y, Urakami T, Aoki Y.
Infectious Disease and Hospital
Epidemiology, Kagoshima Seikyou Hospital, Japan. looking_for_the_true_adoration@yahoo.co.jp
Intern Med 2017;56(5):487-491.
Abstract: Objective:
Legionnaires' disease (LD) is a common form of lobar pneumonia, but the optimum
diagnostic modality has long been a subject of debate due to incomplete
sensitivity and specificity. A delay in the initiation of specific therapy for
LD is associated with increased mortality. The decision to treat a patient for Legionella
must be made quickly. The purpose of this study was to evaluate the ability of
the modified Winthrop-University Hospital WUH system to identify LD while
discriminating against pneumococcal pneumonia at the time of hospitalization for
community-acquired pneumonia. Methods: Five patients with LD and 13 patients
with pneumococcal pneumonia were retrospectively analyzed. Results: The WUH
system identified 4 of 5 patients with LD (sensitivity, 80%) while excluding
legionellosis in 12 of 13 patients with pneumococcal pneumonia (specificity,
92%). The positive and negative likelihood ratios were 10.4 and 0.2. The area
under the receiver operating characteristic curve was 0.969. Conclusion: The WUH
system is useful for obtaining a rapid presumptive clinical diagnosis of LD.
Further investigation with a larger number of patients is strongly recommended.
D'Angelo
A, De Simone C, Pagnottella
M, Rossi S, Pepe R, Ruggieri G, Cocco G, Schiavone C.
UOSD Ecografia Internistica, Università degli studi G.
d'Annunzio Chieti-Pescara, Chieti, Italy. alessio.dangelo@libero.it
J Ultrasound 2017
Feb;20(3):243-245.
Abstract: A 36-year-old man was admitted to the
emergency department of "SS Annunziata" hospital in Chieti complaining
of a sharp chest pain arisen some hours before admission. On examination, the
patient looked sweaty; his vital signs showed tachycardia and augmented breath
rate; sinus tachycardia and normal ventricular repolarization were observed on
ECG, and no abnormalities were observed in the echoscan of the hearth. According
to the clinical and electrocardiographic findings, and to previous episode of
DVT in anamnesis, a thorax CT scan was performed in order to rule out pulmonary
embolism. It showed an "area of parenchymal consolidation involving almost
all the left lower lobe with patent bronchial structures"; given the
patient's CURB 65 score, he was then admitted to the pneumology ward where
empiric treatment with levofloxacin (750 mg PO once daily) was initiated.
Thoracic ultrasound was performed using a multifrequency convex transducer, and
the posterior left area was examined through intercostal approach, placing the
patient in a sitting position. A subpleural patchy hypoechoic lesion with
irregular boundaries was detected; the maximum diameter was 11 cm, and the
multiple hyperechoic spots inside it (elsewhere defined as "air bronchogram")
showed no Doppler signal. Given the positive result of the Legionella
urinary antigen test, antibiotic treatment was switched to Levofloxacin 1000 mg
PO once daily and Claritromicin 500 mg PO twice daily. After 3 days, his
clinical conditions improved dramatically. Ultrasound performed after 5 days
from the diagnosis showed decreased dimensions of the lesion previously
identified (maximum diameter 8.25 cm) and a marked reduction of the hyperechoic
spots in it. The patient was discharged in good clinical conditions, and both
thorax CT scan obtained after 1 and 4 months from the diagnosis showed
radiological resolution of the parenchymal consolidation. The key to ultrasound
visualization of pneumonia is its contact with the pleural surface (86-98% in
cases of CAP) and the relative loss of aeration of the portion involved by the
infection and a concomitant increase in the fluid content. A paradigmatic US
image for parenchymal inflammatory infiltrate has not been established yet;
anyway, some typical findings, when combined with the clinical features, can
confirm the diagnostic hypothesis.
Huang
WC, Tsai HC, Tao CW, Chen JS, Shih YJ, Kao PM, Huang
TY, Hsu BM.
Department of Earth and Environmental Sciences,
National Chung Cheng University, Chiayi, Taiwan, ROC. bmhsu@ccu.edu.tw
PLoS One 2017
Feb;12(2):e0170992.
Abstract: In this study, we describe a nested PCR-DGGE strategy to detect Legionella
communities from river water samples. The nearly full-length 16S rRNA gene was
amplified using bacterial primer in the first step. After, the amplicons were
employed as DNA templates in the second PCR using Legionella specific
primer. The third round of gene amplification was conducted to gain PCR
fragments apposite for DGGE analysis. Then the total numbers of amplified genes
were observed in DGGE bands of products gained with primers specific for the
diversity of Legionella species. The DGGE patterns are thus potential for
a high-throughput preliminary determination of aquatic environmental Legionella
species before sequencing. Comparative DNA sequence analysis of excised DGGE
unique band patterns showed the identity of the Legionella community
members, including a reference profile with two pathogenic species of Legionella
strains. In addition, only members of Legionella pneumophila and
uncultured Legionella sp. were detected. Development of three step nested
PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella
community. The method is rapid and provided sequence information for
phylogenetic analysis.
Farzi N, Abrehdari-Tafreshi Z, Zarei O, Chamani-Tabriz L.
Reproductive Biotechnology Research Center, Avicenna
Research Institute (ACECR), Tehran, Iran. lchamani@gmail.com
Int J Hematol
Oncol Stem Cell Res 2017 Jan;11(1):49-53.
Abstract: Background: Legionella
pneumophila (L. pneumophila) is a gram-negative bacterium which
causes Legionnaires'
disease as well as Pontiac fever. The Legionella infections in patients
suffering from neutropenia- as a common complication of cancer
chemotherapy- can distribute rapidly. We aimed to detect of L. pneumophila in
hematological malignancy suffering patients with neutropenic
fever by targeting the (macrophage infectivity potentiator) mip gene. Subjects
and Methods: Serum and urine specimens were obtained from 80 patients and
presence of mip gene of L. pneumophila in specimens was investigated by PCR. Results: The
L. pneumophila infection was detected in 21 (26.2%) and 38 (47.5%)
of urine and serum specimens, respectively. Conclusion: Our findings
indicated that the relative high prevalence
of L. pneumophila in the studied patient’s group which show the
necessity of considering this
microorganism in future studies from detection and treatment point of view in cancer patients.
