Testing for Legionella spp

Background

• First recognized outbreak of Legionnaire’s Disease was at an American Legion Convention in Philadelphia in 1976

• Within one week, approximately 130 people were hospitalized due to exposure and 25 died. In total, 34 people died of the disease.

• CDC determined source to be the cooling tower of the hotel’s air conditioning system

• Efforts of the CDC led to the first time the organism was cultured and identified

Transmission

• Contaminated, aerosolized water from sources become airborne

• Infection occurs when a host inhales the contaminated aerosol

• Disease can occur in susceptible hosts• Immunocompromised

• Heavy drinking/heavy smoking

• Age

Transmission

• Legionella is ubiquitous in all aquatic environments. • Present in ground water, freshwater, and marine waters (sources of the

organism in plumbing systems, spas, cooling towers, etc)

• Elimination and removal is not possible and control measures are required to prevent stagnation of water and accumulation of bacteria

• Routine monitoring of Legionella levels are recommended to evaluate trends in Legionella concentration and to validate specific water safety procedures

• Legionella can be difficult to treat and eliminate because it can survive in protozoa and biofilms which aid in protection

Legionnaire’s Disease

• Severe form of pneumonia• High fever; severe, productive cough; nausea, vomiting, stomach

discomfort; diarrhea• Other symptoms include headaches, muscle aches, chest pain,

shortness of breath• Despite treatment, some individuals will suffer long-term effects of the

disease which include persistent fatigue, neurological symptoms, neuromuscular symptoms

Pontiac Fever

• Acute, mild upper respiratory infection

• Flu-like symptoms with no pneumonia

• Resolves on its own within 2 - 5 days

• Caused by the same bacteria that causes Legionnaire’s disease

Legionella in Industrial facilities

• Legionnaire’s disease linked to petrochemical plant• Pas-de-Calias, France: Nov. 2003-Jan. 2004, 86 cases (18 deaths) (Tran Minh et al. JID 2006:193)

• Investigation of waste water treatment plants • 2006 Finnish study: 2 cases of Legionnaire’s disease due to high levels of Legionella

pneumophila at the plant (up to 1.7 × 1010 cells/l; Kusnetsov et al. BMC Infectious Diseases 2010, 10:343)

• Survey of 25 US drinking water treatment plants • Demonstrated Legionella pneumophila in 25% of source water and 4% in treated water (King et

al., Sci Total Environ 2016, 562: 987)

Legionella spp

• Gram negative, facultative, intracellular bacteria

• Commonly found in soil and water

• Slow-growing

• More than 50 species identified, at least 18 associated with human disease

• >90% of cases of Legionnaire’s disease in Europe and in the US are caused by Legionella pneumophila

Legionella testing

• Culture method

• ELITE (Environmental Legionella Isolation Techniques Evaluation) program of CDC certifies laboratories proficient in isolating and identifying Legionellathrough culture- based method

• Labs receive 2 panels of unknowns per year

• Labs that correctly identify the panel which is positive for Legionella are given the certificate of proficiency and are listed as ELITE members

Media used for cultures

Buffered Charcoal Yeast Extract (BCYE) BYCE + with cysteine and BYCE – without cysteine

PVC (Polymyxin B, vancomycin, cycloheximide) BCYE media with antibiotics and with and without cysteine

Inhibits secondary flora

GPVC = PVC media with glycine Addition of glycine also inhibits secondary flora from growing

Culture Method

• Concentrate 1 liter by filtration using a 0.22um polycarbonate filter

• Vortex the filter with 5ml of sterile water for 1 minute

• Treat 1ml of sample with acid buffer

• Pipette 0.1ml of sample on to the following media (BYCE,PCV,GPCV)

Culture Method

• Suspect colonies on these plates (BYCE, PCV, GPCV) are grown on the L-cysteine negative plates

• If no growth on the cysteine negative plates after 10 days incubation, then colonies are presumptive for Legionella

• Suspect colonies may also demonstrate fluorescence

Ideal growth of cultures in enriched and selective media

BCYE PCV

GPCV PCV without cysteine

BCYE PCV GPCV

Typical bacterial growth from wastewater treatment plant samples

Use of Assays for the determination of Legionella presence in samples

• Culture

• How to determine presence of viable cells?Day 1 Day 7

Limitations of Culture

• Long assay time

• Low sensitivity (can be as low as 50-60%)

• Loss of viability after collection or sample treatment

• Presence of interfering microbiota

• Inability to detect viable but non-culturable state and cells within amoeba

qPCR for Legionella

• Amplification of DNA is monitored at every cycle using fluorescent reporter dye

• Amount of fluorescence is directly proportional to amount of DNA in sample

• DNA is quantified during the exponential phase• Many different types of probes can be employed for quantitative

PCR• Employ double-dye probes (TaqMan probes)

• Probes and primers specific for • Legionella sp., Legionella pneumophila, and Legionella pneumophila serotype 1

qPCR for Legionella• Standard curves generated from

known amounts of DNA to

compare against unknowns

qPCR standard curves

y = -3.3912x + 38.085R² = 0.9992

0

5

10

15

20

25

30

0 2 4 6 8

Legionella spp. STD Curve

Log copy number

Mea

n C

t

y = -3.2709x + 39.167R² = 0.9998

0

5

10

15

20

25

30

35

40

0 2 4 6 8

L. pneumophila 16S STD Curve

Log copy number

Mea

n C

t

y = -3.2566x + 39.778R² = 0.9905

0

5

10

15

20

25

30

35

40

0 2 4 6 8

L. pneumophila SG1 STD Curve

Log copy number

Mea

n C

t

Advantages vs Disadvantages of qPCR

• Advantages• Highly sensitive and quantitative

• Short TAT

• Good specificity using multiple primers and probes

• Has been suggested for monitoring of water samples

• Disadvantages• No discrimination between live and dead

cells• DNA can persist in the environment for a long

period of time after cell death

• Possible inhibition of PCR reaction due to compounds that can prevent amplification

• Not officially accepted like the culture method; no guidelines on testing and interpretation

HybriScan

• Developed by ScanBec, distributed by Sigma Aldrich

• Rapid molecular assay targeting rRNA of Legionella sp.

• Uses specific capture and detection probes for the rRNA target – sandwich hybridization

• Detection probe binds to an ezyme that yields a color reaction

• Intensity of color proportional to the amount of cells

HybriScan

Overview

Overview

Overview

Standard cells/well Mean Value1 0 0.0862 1000 0.183 3000 0.3314 10000 0.945

Advantages of HybriScan

• Rapid – without enrichment, can take half a day

• Inexpensive – large number of samples per plate

• High sensitivity (high levels of rRNA in living organisms as opposed to 1 DNA molecule)

• High specificity (2 probes used for specific, conserved sites in rRNA)

• Qualitative and quantitative analysis of living cells only (rRNA is fast decomposed in dead cells)

Disadvantages of Hybriscan

• No differentiation of serotypes or subspecies

• Limited probe design (target rRNA only)

• Not officially accepted for Legionella testing; no guidelines for testing and interpretation

Comparison Studies

• CDC Legionella sample serially diluted in 10mL sterile water• All dilutions plated onto PCV media and colonies counted• DNA extracted from 0.60mL of each suspension for qPCR• 3.4mL of each suspension used for extraction of RNA for the HybriScan

assay• qPCR and HybriScan used for CDC ELITE samples for comparison to

culture method

Comparison studies – culture on PCV

Comparison studies – culture vs. qPCR

y = 0.9443x + 2.2306R² = 0.9894

0.01.02.03.04.05.06.07.08.09.0

10.0

0.0 2.0 4.0 6.0 8.0

CFU/mL vs. Legionella spp qPCRLo

g co

pies

/m

L

Log cfu/mL

y = 0.9664x + 2.0311R² = 0.986

0.01.02.03.04.05.06.07.08.09.0

0.0 2.0 4.0 6.0 8.0

Log cfu/mL

Log

copi

es /

mL

CFU/mL vs. L. pneumophila 16S qPCR

y = 0.9239x + 2.0166R² = 0.991

0.01.02.03.04.05.06.07.08.09.0

0.0 2.0 4.0 6.0 8.0

CFU/mL vs. L. pneumophila SG1 qPCR

Log cfu/mL

Log

copi

es /

mL

Comparison studies – HybriScan vs. qPCR or culture

y = 0.314x + 2.5292R² = 0.851

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 5.0 10.0

HybriScan vs. qPCR Legionalla spp.

Log copies/mL

Log

cells

/mL

y = 0.3006x + 2.6454R² = 0.8699

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 5.0 10.0

HybriScan vs. qPCR L. pneumophila 16S

Log copies/mL

Log

cells

/mL

y = 0.3226x + 2.5733R² = 0.8585

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0 5.0 10.0

HybriScan vs. qPCR L. pneumophila SG1

Log copies/mL

Log

cells

/mL

y = 0.3023x + 3.2178R² = 0.9289

2.5

3.0

3.5

4.0

4.5

5.0

5.5

0.0 2.0 4.0 6.0 8.0

CFU/mL vs. cells/mL (HybriScan)

Logs Cells/mL

Log

CFU

/mL

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

10^0

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

LOG CFU/ML ON PCV

LOG QPCR - LEG SPP

LOG QPCR - LPN 16S

LOG QPCR - LPN SG1

LOG HYBRISCAN -QUANTITATIVE

PEARSON'S CORRELATION

CultureqPCR -

Legionella spp

qPCR - L. pneumophila

16S

qPCR - L. pneumophila

SG1 HybriScan

Culture 1.00 0.96 0.97 0.97 0.97

HybriScan 0.97 0.90 0.92 0.91 1.00

CDC PT samples

CDC SAMPLE

CULTURE POSITIVE

OR NEGATIVE

QPCR - Legionella

spp. (copies/mL)

QPCR - Legionella

pneumophila 16S

(copies/mL)

QPCR - Legionella

pneumophila SG1

(copies/mL)HybriScan - Qualitative

HybriScan - Quantitative

(cells/mL) ACTUAL RESULTS CDC SPECIES MALDI-TOF ID CDC CFU/mL

1 NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NA NEGATIVE NA None Identified NA2 POSITIVE 1.90E+07 NEGATIVE NEGATIVE POSITIVE 84902.6 POSITIVE L. gormanii Legionella gormanii 13 POSITIVE 1.60E+10 1.50E+10 NEGATIVE POSITIVE 144300.1 POSITIVE L. pneumophila Legionella pneumophila 10004 NEGATIVE 9.70E+07 1.20E+08 7.50E+07 POSITIVE 144300.1 POSITIVE L. pneumophila No Growth 35 POSITIVE 1.90E+07 1.40E+08 1.50E+08 POSITIVE 144300.1 POSITIVE L. pneumophila Legionella pneumophila 1446 NEGATIVE 0.00E+00 NEGATIVE NEGATIVE NEGATIVE NA NEGATIVE NA None Identified NA

Southerly Wastewater Treatment Plant

Site 1 Site 2 Site 3

Westerly Wastewater Treatment Plant

Site 1 Site 2 Site 3

Westerly Legionella sample

LOCATION

Culture (log

cfu/mL)

qpCR Leg spp

log copies/m

l

qPCR 16S log

copies/ml

qPCR SG1 log

copies/mlHybriScan Qualitative

HybriScan Quantitative log cells/ml

MALDI-TOF

Maintenance building

cooling tower feed 0.0 0.0 0.0 0.0 NEGATIVE 0.0 NEGATIVE

Maintenance building

cooling tower drain 1.8 2.6 1.6 0.0

QUESTIONABLE 3.5

Legionella anisa

Maintenance building

cooling tower reservoir 2.0 2.7 1.3 0.0

QUESTIONABLE 3.7

Legionella anisa

How to report?

• Culture is the gold standard; however, screening and confirmation can be done on samples using alternative method

• Cultures negative, qPCR and HybriScan positive for Legionella• A positive result cannot be ruled out due to the overgrowth demonstrated which can prevent growth

of Legionella

• HybriScan results can demonstrate the presence of viable Legionella sp. in the samples and qPCR can confirm if any isolated L. pneumophila is serogroup1.

• Further studies with MALDI will determine identity of contaminants• Bacillus sp., Brevibacillus formosus, Rhodococcus sp., Mycobacterium chelonae, Acinetobacter johnsonii, Legionella anisa

Microcystin survey

• Cooperative Project Funding Agreement (PFA): 04647, Titled the Evaluation and Optimization of Cyanotoxin Analytical Methods.

• Goal of survey = to compile information on the use of ELISA and LC/MS/MC and quality control information for testing microcystin

• Survey ELISA Methods• https://www.surveymonkey.com/r/WRF-ELISA-04647

• Survey LC/MS/MS Methods• https://www.surveymonkey.com/r/WRF-04647-LCMSMS