Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment

The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires’ disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires’ disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10–14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at>1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2020.004 ://iwaponline.com/jwh/article-pdf/18/3/345/759666/jwh0180345.pdf Katherine E. Fisher (corresponding author) Leah P. Wickenberg William F. McCoy Phigenics Research and Innovation Team, Nevada Center for Applied Research, Reno, NV, USA E-mail: kfisher@phigenics.com Lesley F. Leonidas Michelle A. Habib Phigenics Analytical Services Laboratory (PASL), Warrenville, IL, USA Anna A. Ranz Rafael M. Buford Phigenics Analytical Services Laboratory (PASL), Fayetteville, AR, USA


INTRODUCTION
Legionella bacteria are opportunistic pathogens that can cause respiratory infections in humans. These infections range in severity from Pontiac fever to Legionnaires' disease (LD). Pontiac fever is less severe with symptoms lasting approximately 1 week and does not develop into pneumonia (CDC a). In contrast, LD is an extreme form of pneumonia, with symptoms lasting weeks, and has the potential to cause long-term debilitating effects including fatigue, memory loss, muscle pain, and post-traumatic stress disorder (Lettinga et al. ). LD can be treated with antibiotics, but has an overall mortality rate of 1 in 10 (CDC b). Legionella enters the respiratory tract via the inhalation of aerosolized water droplets from contaminated water or by aspiration. The detection of these bacteria before infection is critical, especially in healthcare buildings with a high risk of exposure for immunocompromised persons (Marston ). Specifications for the actionable concentration  A negative predictive value (NPV) describes the ratio of PCR-negative results to culture-negative results; the higher the NPV the more reliable the screen is. The goal of this study was to quantitatively determine the NPV of the Next Day Legionella PCR in comparison with the ISO 11731 culture method and to assess its effectiveness as a negative screen for validation testing in WMPs.

Sample collection
Eleven thousand one hundred and twenty-five (11,125) water samples were collected across the United States for

DNA extraction: Method 2
After the main study was completed, a 2-month trial was done with an improved DNA extraction method to increase the NPV. Method 2 was used on the final 2,012 samples. The new method was as follows: 2 ml of the potable water concentrate or 2 ml of the non-potable water sample was pelleted by centrifugation and 2 ml of the supernatant was discarded. The remaining pellet was used for DNA extraction with the proprietary P.U.R.E.™ method. The PCR and the culture method remained the same.
Laboratory experiment comparing DNA extraction methods A laboratory experiment was performed by the Phigenics Research and Innovation Team to compare Methods 1 and 2. Three Legionella isolates, one L. pne sg 1, 2-14, and species, were diluted to OD 600 0.1 and serial diluted to 10 À5 . The three lowest dilutions were used in the experiment and 100 μl of each cell suspension was diluted in 9.9 ml of PBS to simulate a 10 ml filter concentrate. Each sample was plated on GVPC (100 and 500 μl) and extracted with Methods 1 and 2. The DNA extracts were analyzed with the Next Day Legionella PCR. The plates were incubated at 35 ± 1 C for 6-10 days. The CFU/ml reported in the results relates to the hypothetical 100 ml sample that the simulated 10 ml filter concentrate came from.

Real-time PCR
An ISO 12869:2012 compliant commercial Legionella realtime PCR kit was used for the Next Day Legionella PCR.
This method detects 21 Legionella species, including L. pneumophila, by targeting a specific sequence of the 16 s rRNA gene. It also contains manufacturer provided quality control measures including an internal control, a positive control and a negative control. Validation for exclusivity against THAB strains is also reported by this method.
Sixteen species of common heterotrophic bacteria were included in the validation. The manufacturer's instructions were followed for reaction set-up, thermocycling and data analysis. In the occurrence of PCR inhibition, the DNA sample was diluted 1:10 and re-analyzed.

Data analysis
The PCR results show a detect or non-detect for Legionella DNA in the water sample. These data were compared with the culture results. The data were grouped by (1)  (4) PCR À /ISOÀ. The NPV and positive predictive value (PPV) of the test were calculated accordingly: Additional statistics of accuracy, specificity and sensitivity were calculated as follows:

Water samples
A total of 11,125 water samples were analyzed in this twopart study. Of these samples, 10,094 (90.73%) were potable and 1,031 (9.27%) were non-potable (mostly cooling tower water samples). There were 10,487 (94.18%) samples col-  Legionella PCR at >10 CFU/ml was 99.95% (Figure 2). At >2 CFU/ml, the NPV was 99.92% and at >1 CFU/ml the NPV was 99.85%. Table 1 shows the NPV for all 9,113 water samples; for potable and non-potable samples, the overall NPV was 99.17% at an LOD of 0.1 CFU/ml. Table 2 was used to calculate the NPV of the Method 1 dataset. A small portion of the samples were invalid (0.37%, n ¼ 34) because the samples were culture-positive but PCR-negative (false-negative).

The contingency table in
The sensitivity of the test was 94.88% due to these falsenegative results ( Table 1). The majority of the invalid results occurred at 1 CFU/ml and originated from 100 ml water samples. Figure 3 shows the percentage of these invalid results out of the total number of culture-positive samples at the same CFU/ml. Twenty-seven samples (15.08% of 1 CFU/ml positives) were invalid at the 1 CFU/ml level, and three samples (3.41%) were invalid at the 2 CFU/ml level. Similarly, one sample (2.82%) and two samples (0.63%) were invalid in the ranges 3-10 and >10 CFU/ml, respectively ( Figure 3).

Method 1 PPV
Legionella DNA was detected in 5,006 (54.90%) samples using Method 1. The PPV was 14.06% for the entire sample set of 9,113 water samples. For potable and nonpotable samples, the PPVs were 15.23% and 7.37%, respectively (Table 1). The accuracy of this test (52.40%) and the specificity of this test (48.63%) were very low, due to the Figure 2 | NPV of the Next Day Legionella PCR. The NPV is shown for each method tested. Method 1 (n ¼ 9,113) consisted of a DNA extraction with a lower sensitivity than Method 2 (n ¼ 2,012). The CFU threshold was used to calculate the NPV at different limits of detection.     Case study: negative screen use in a chloraminated system The monthly trend of water sampling results from a healthcare facility over the 9-month period is shown in Figure 5 (n ¼ 1334 samples). This example consisted of 4% nonpotable and 96% potable water samples. Overall, there was an upward trend in both PCR-positive and culturepositive samples from April to July, and a downward trend from July through November as the weather got warmer and colder, respectively. The PCR-positive rate was on average 40% higher than the culture-positive rate throughout the year. This facility maintained water quality with free residual chlorine until December when chloramination was implemented. Figure 5 shows that in December, the PCR-positive rate was 96%, approximately 50% higher    than the average of the 8 months before and 80% higher than November (16% PCRþ). Additionally, after chloramination was implemented, water quality degradation was observed as indicated by higher THAB counts, higher free ammonia concentrations and detections of Mycobacterium in more than 50% of samples (data not shown). However, after chloramination was implemented, the Legionella culture-positive rate dropped to 0% in December from 0.79% in November and 9.6% in October. Chloramination of potable water has been associated with inducing the VBNC state in

Water samples
The majority of the water samples in this study were potable and collected from healthcare facilities. This type of sampling location is in high proximity to at-risk patients; therefore, there is an increased risk of acquiring LD if there is Legionella contamination in these building water systems. It is important to maintain clean and pathogenic bacteria-free water in the healthcare setting through the use of a WMP that includes ver-  Originally, there were 38 invalid results, but 4 of these isolates were incorrectly identified as Legionella by the traditional culture method. These isolates were presumed to be Legionella after culturing, testing cysteine auxotrophy, and performing the latex agglutination test. Speciation by DNA sequencing was performed and the assay was negative for any Legionella spp. This shows that the negative screen PCR-negative result was correct. These results confirm inherent limitations in the traditional culture method.
There is a need for genomic analysis of Legionella from validation testing due to situations like this. The PCR results can be confirmed through genomic analysis, so that isolate con- It is possible to determine these groups with more specific PCR, but genomic analyses can provide more accurate information such as the exact species of Legionella or the sequence type of L. pneumophila. Genomics will greatly improve the defensibility of a WMP if there is a case of disease. Additionally, genomics will allow the WMT to take specific action depending on the virulence of the pathogen.
The CDC speciates all Legionella isolates via the mip gene PCR and the European Study Group for Legionella infections has built a database for L. pneumophila sequence types. The Legionella spp. negative screen should be the first step in the validation of Legionella management in building water. This diagnostic has allowed facility managers to decide that it was safe to return water recirculation loops back into commission the day after remediation. Results from PCR negative screening provide a focused approach to culturing and verifying Legionella isolates.

Method 1 PPV
The PPV for the Legionella negative screen was quite low at 15.23% potable and 7.37% non-potable for the 9,113 sample set (Table 1). This means that the probability is very low that PCR-positive results will correlate to culture-positive results.
There are many reasons for this discrepancy, one being the sensitivity of the PCR method. The culture method has a A novel viability assay is needed that possibly brings together the sensitivity of PCR and the ability to differentiate live and dead cells.

Method 2 provides increased sensitivity
Due to the fact that the NPV was less than 100%, there was room for improvement in this method. One area of optim-  (Figure 2).
The laboratory experiment (Figure 4)  High PCR-positive rates in non-potable systems The PPV for non-potable samples was 6.5% lower than for potable samples. This indicated that non-potable samples are more likely to be PCR-positive, culture-negative. A significant portion of the non-potable samples, however, were PCR-negative. A PCR-negative result in non-potable water is indicative of Legionella-free water and is very useful information for the WMT. A PCR-positive, culture-negative result could be due to dead cells in properly treated cooling tower water disinfected with much more toxic antimicrobials than potable water. In addition, non-potable water sources are likely to have much more organic matter than potable water. This has been shown to decrease the effectiveness of chlorine disinfectants (Virto et al. ). In this situation, a VBNC state or a ghost cell state can occur, thus increasing the PCR-positive rate and lowering the culture-negative rate.
In the non-potable water case study shown in Figure 6, the PCR-positive rate was 40% higher than in the healthcare facility study, where the majority of samples were potable ( Figure 5). This non-potable water system may have had VBNC cells or ghost cells that the Next Day Legionella PCR detected but cannot differentiate; therefore, a viability assay is particularly important for non-potable water. There are many benefits to the negative screen strategy, but the inability to differentiate viable cells is a limitation.
The Next Day Legionella PCR is a foundational test that can be built upon for future Legionella diagnostics including more accurate viability assays.

COMPETING INTERESTS STATEMENT
During this research study, all authors were employed by Phigenics, LLC.

FUNDING INFORMATION
Funding for this research was provided by Phigenics, LLC.
helpful suggestions and comments on the manuscript. We also give special thanks to Dr. Claressa Lucas (CDC) for her feedback and expert suggestions for the manuscript.