MOLECULAR
Methods
Increasing Use of PCR and qPCR Spurs New Accreditation from AIHA-LAP
BY ED RUTKOWSKI
Knowing the sequence of DNA’s building blocks allows laboratories to develop highly specific tests that target unique regions of genetic code.
RESOURCES AIHA: Recognition, Evaluation, and Control of Legionella in Building Water Systems (May 2015).
AIHA-LAP, LLC: “Environmental Microbiology Laboratory Accreditation Program (EMLAP)” (November 2016).
CDC: “Vital Signs: Deficiencies in Environmental Control Identified in Outbreaks of Legionnaires’ Disease—North America, 2000–2014” (June 2016).
Public Works and Government Services Canada: MD15161-2013: Control of Legionella in Mechanical Systems, Appendix D: Legionella Testing Protocols (July 2015)
Saleh, Mike: “Molecular Methods Training Session” (webinar conducted April 2017).
As Saleh explained in the webinar, qPCR is typically used for Legionella, food testing (for Salmonella, E. coli, and Listeria), clinical testing (for sexually transmitted diseases), and diseases such as Lyme disease and rabies. qPCR also figures in EPA’s Environmental Relative Moldiness Index (ERMI), a method that quantifies 36 molds that can be present in the indoor environment.
In qPCR, a lab analyst mixes DNA extracted from the sample with a cocktail of chemicals, including the enzyme polymerase; stray nucleotides; fluorescent molecules known as “probes”; and short bits of single-stranded DNA called “primers,” which are prepared to target the organism of interest.
Next, the analyst inserts tubes containing the mix into a machine known as a thermal cycler, which heats, cools, and reheats the mix to specific temperatures, allowing temperature-dependent reactions to occur. At 94–98 degrees Celsius, the double-stranded DNA from the sample splits in two. When the cooling cycle lowers the temperature to 54–60 C°, the primers attach to target regions of DNA, if present. Heating again to 72–80 C° causes the polymerase to bind the stray nucleotides to the DNA regions adjacent to the primers, completing the cycle and doubling the DNA, a result known as “amplification.” Typically the machine will run for approximately 40 cycles, producing millions of copies of DNA in a few hours. In qPCR, each reaction that doubles DNA releases the probes. The machine detects the presence of these fluorescent molecules, allowing the quantitation to occur.
Conventional PCR proceeds similarly to qPCR, with some significant differences. Probes are not used in conventional PCR, and the primers can be either “universal” or “specific,” depending on the purpose of the analysis. A universal primer will be used if the lab is trying to determine whether a sample contains any bacteria or any mold. If the purpose is to detect the presence of a specific bacterium (Legionella, for example) or a specific mold (Aspergillus flavus, say) the lab will use specific primers designed to attach to the relevant portions of DNA.
Conventional PCR also requires some additional steps, including application of a technique known as gel-electrophoresis, which confirms that amplification occurred. If the lab has used a specific primer, the analysis could end with this step. For barcoding (the identification of an unknown organism, which involves use of universal primers), an additional step produces a chromatograph of the genetic sequence—the nucleotide base pairs—which were amplified during the thermal cycling. Saleh says that labs often outsource this step since it requires an expensive piece of equipment. Finally, to identify the sequence, labs will paste it into one of several databases of genetic data. GenBank, the oldest such database and, according to Saleh, by far the largest, is typically used for this purpose. SLOW ACCEPTANCE Labs have used PCR and qPCR to test for foodborne pathogens for decades, but PCR analysis has been adopted for industrial hygiene purposes only recently. Sobek attributes this delay to several factors.
“We were talking PCR back in late 2004, 2005, and it’s been on the radar with industrial hygiene, but the acceptance has been really, really slow,” Sobek says. “Obviously, one of the drivers of that is price point.”
Most of Sobek’s industrial hygiene customers at Assured Bio are interested in analysis of mold samples. He says that IHs accustomed to $25 spore trap analyses can experience sticker shock when considering PCR, whose costs typically range from $100 to $200 per sample.
Education has also played a role as IHs become familiar with the more sensitive analysis PCR provides. “The detection level is so much greater,” Sobek says of PCR. “Your samples look really different, and you’re seeing a lot more of something than you ever saw before in the same environments you’ve been collecting things from. That’s a difficult thing to absorb and deal with. It takes time.”
In Canada, use of PCR for IH purposes has been impelled by a standard issued in 2016 that requires qPCR tests for Legionella in the cooling towers of federal buildings. According to Appendix D of Control of Legionella in Mechanical Systems, qPCR tests must be performed at least weekly during Legionella emergencies and within 24 hours of start-up cleaning and disinfection. qPCR is also required if, following a cleaning and disinfection, culturing indicates a Legionella concentration greater than 10 colony-forming units per milliliter (cfu/ml) or a qPCR test indicates L. pneumophila in excess of 100 genome equivalents per milliliter (ge/ml).
The standard “took us by surprise,” says Rafic Dulymamode, laboratory manager at Pinchin Ltd in Mississauga, Ont. “All the labs had to scramble to get [qPCR] added as a test.”
Pinchin, which has also earned AIHA-LAP accreditation for the PCR FoT, recently used PCR and qPCR to help a large institution with many buildings manage a Legionella problem. Dulymamode says this experience shows how PCR and qPCR can be used to contain Legionella without severely disrupting business functions. “A routine test of domestic hot water systems showed that there was a high level of Legionella pneumophila by the culturing method,” Dulymamode explains. Hot water to buildings was shut down and reopened in sequence following disinfection and confirmation that Legionella were absent. “By efficiently using a mixture of qPCR and culturing, we have helped this institution manage the situation with minimal impact on its activities,” Dulymamode says.
Jason Dobranic, vice president of microbiology and life sciences at EMSL Analytical, says that some of his lab’s clients are using PCR in a similar manner. “They might take a sample for PCR and then a sample for culture at the same time, but we’ll do the PCR in 24 hours and they’ll at least start to pinpoint where there might be problems,” Dobranic says. “Say it’s in a hospital, and they found one water pipe or outlet that’s positive for Legionella. They could at least tell people, ‘Don’t use this,’ and maybe start looking at remediation of that area. You can do that right away, within a day. And you can just follow up with the culture.” SAMPLING AND PCR EMSL started using PCR in 2001 and has developed a variety of tests for molds, bacteria, and food pathogens, Dobranic says. Some EMSL clients submit food samples for authentication: they might be shipping in tilapia from China, for example, and will want PCR to verify that the white meat in the containers is the intended product. EMSL also does PCR tests for bed bugs, fecal indicators, and, of course, Legionella.
Asked whether he has any advice for his industrial hygiene clients about PCR analysis, Dobranic stresses the importance of communicating with the laboratory prior to sampling to verify the proper media. “A lot of times, they’re specialized cassettes,” Dobranic says. “If you’re doing an air sample, [the cassettes] have to be not only sterile but DNA free. They have to be manufactured under strict conditions so they’re compliant with a PCR-type sample.”
Sobek has similar advice for industrial hygienists who intend to submit samples for PCR analysis. “You’ve got to be really careful,” he says. “You’ve got such great specificity now and such great detection levels, you’ve got to make sure you don’t cross-contaminate your samples.” For some clients, Sobek says, PCR results will show a lot Stachybotrys in samples from one location and much smaller amounts in samples from other locations. “We’ve gone back and retested those locations, and we don’t find it,” he says—an indication that the mold from the first location has found its way into samples from subsequent locations. IHs should be sure to change gloves and Tyvek suits before moving to another site, Sobek says.
“Inhibition is a problem with PCR” for Legionella, says Dulymamode. The thermal cycler “is such a sensitive piece of equipment, and dirt and residues of chemicals tend to prevent the system from working well. So the quality of the sample is very important.”
Despite these complications, Dulymamode advocates greater use of PCR and anticipates that it will eventually become the go-to method for control of Legionella, and possibly for other hazards.
“We still have to make sure that this test is used more widely, especially for Legionella testing,” he says. “This is how I see the future for Legionella testing, that qPCR will become a screening tool and culturing will just be a confirmatory test for all those positives that have been found through qPCR.” ED RUTKOWSKI is editor in chief of The Synergist. He can be reached at (703) 846-0734 or via email. The Synergist gratefully acknowledges the assistance of Michael Saleh, Rafic Dulymamode, Jason Dobranic, and Ed Sobek in the development of this article.
In June 2015, an outbreak of Legionnaires’ disease in the Bronx section of New York City sickened 128 people and led to 12 deaths. It was the worst outbreak of Legionnaires’ in the city’s history, and one of the deadliest in the United States since the first recognized incidence of the disease, at the eponymous 1976 Legionnaires’ convention in Philadelphia. Public health authorities linked the Bronx outbreak to five cooling towers at a hospital and two hotels in the borough. The Bronx outbreak is part of an alarming trend. According to a CDC summary of outbreaks in North America, between 2000 and 2014 the incidence of Legionellosis, which includes both Legionnaires’ disease and Pontiac Fever, increased 286 percent, from 0.42 to 1.62 per 100,000 persons. These numbers are likely lower than the true incidence, since investigations of Legionnaires’ require an environmental assessment, which isn’t typically performed for cases that aren’t part of a recognized outbreak. One reason for Legionnaires’ high fatality rate—10 percent of all cases result in death, according to CDC—is the amount of time required to determine the source of an outbreak. Laboratories typically use a culture method to confirm the presence of Legionella pneumophila bacteria, the species that causes most cases of Legionnaires’ disease, in a water-based sample. But L. pneumophila grow slowly in culture, and results typically require 10 to 14 days, delaying remediation and increasing the likelihood that more people will be exposed. Increasingly, though, laboratories are using molecular analytical techniques that identify bacteria like L. pneumophila much more quickly. Polymerase chain reaction, or PCR, is an analytical method that amplifies DNA in a sample and allows identification of a pathogen in a matter of hours. “PCR can be done a lot faster, and a lot more sensitively just based on the fact that sometimes with culture you get things dying in transit,” says Michael Saleh, the laboratory manager at Sporometrics in Toronto. This summer, Sporometrics was one of the first labs to be accredited under a new PCR field of testing (FoT) offered by AIHA Laboratory Accreditation Programs, LLC. During the 2015 Bronx outbreak, the bacteriology laboratory at the Wadsworth Center, which is affiliated with the New York State Department of Health, used PCR to analyze samples from seventeen cooling towers. Within 48 hours, building managers whose towers tested positive for Legionella were able to begin remediation, likely preventing further spread of the disease. However, both regulations and best practice still require culturing for Legionella, which labs typically perform according to ISO 11731:1998, Water quality—Detection and enumeration of Legionella, and the CDC publication Procedures for the Recovery of Legionella from the Environment. PCR is unable to distinguish between dead cells and living, so culturing is necessary to unequivocally confirm the presence of viable bacteria in a sample. Another advantage of culturing is that it allows public health authorities to link particular cases of disease with specific sources by comparing the cultured Legionella isolates at the strain level. According to the managers of several labs accredited by AIHA-LAP, the quick turnaround of PCR results makes it an attractive choice for industrial hygienists and other clients. And PCR’s usefulness extends beyond Legionella and other bacteria. A handful of labs offer PCR tests for a variety of molds, viruses, fungi, fecal indicators, and other hazards. “It’s pretty much unlimited what your targets can be with PCR,” says Ed Sobek, founder and president of Assured Bio Labs in Oak Ridge, Tenn., which has applied for AIHA-LAP accreditation in PCR. “If somebody’s interested in something, it’s pretty easy to whip up the assay, calibrate it, and get it going for the client. It’s very versatile in being able to cover the gambit of microbes that we’re dealing with.” CONVENTIONAL VERSUS QUANTITATIVE PCR owes its versatility to the field of genetic sequencing. Knowing the sequence of DNA’s building blocks—the nucleotide base pairs adenine-guanine and cytosine-thymine, whose precise order determines whether an organism is a Legionella bacterium, an H1N1 virus, a spore of Aspergillus flavus, or a human being—allows laboratories to develop highly specific tests that target unique regions of genetic code. A PCR test for fungi will target what is known as the ITS region of DNA; a test for bacteria will focus on the 16S region. There are two types of PCR analysis. The conventional method, known simply as PCR, is useful for identifying unknown organisms from a pure culture, a process known as “barcoding.” Conventional PCR will tell you what’s in the sample, but nothing else. If a client has a mixed sample and wants to know whether it contains a specific organism, the laboratory will use the quantitative variant of PCR, known as qPCR. “qPCR shines when you’re looking for something specific and you want to quantify it in a sample,” Saleh says. In April, Saleh, who serves on AIHA-LAP’s Analytical Accreditation Board, presented a webinar about PCR to an audience of site assessors to prepare them for visits to laboratories applying for accreditation in the PCR FoT.
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Although the print version of The Synergist indicated The IAQ Investigator's Guide, 3rd edition, was already published, it isn't quite ready yet. We will be sure to let readers know when the Guide is available for purchase in the AIHA Marketplace.
 
My apologies for the error.
 
- Ed Rutkowski, Synergist editor
Disadvantages of being unacclimatized:
  • Readily show signs of heat stress when exposed to hot environments.
  • Difficulty replacing all of the water lost in sweat.
  • Failure to replace the water lost will slow or prevent acclimatization.
Benefits of acclimatization:
  • Increased sweating efficiency (earlier onset of sweating, greater sweat production, and reduced electrolyte loss in sweat).
  • Stabilization of the circulation.
  • Work is performed with lower core temperature and heart rate.
  • Increased skin blood flow at a given core temperature.
Acclimatization plan:
  • Gradually increase exposure time in hot environmental conditions over a period of 7 to 14 days.
  • For new workers, the schedule should be no more than 20% of the usual duration of work in the hot environment on day 1 and a no more than 20% increase on each additional day.
  • For workers who have had previous experience with the job, the acclimatization regimen should be no more than 50% of the usual duration of work in the hot environment on day 1, 60% on day 2, 80% on day 3, and 100% on day 4.
  • The time required for non–physically fit individuals to develop acclimatization is about 50% greater than for the physically fit.
Level of acclimatization:
  • Relative to the initial level of physical fitness and the total heat stress experienced by the individual.
Maintaining acclimatization:
  • Can be maintained for a few days of non-heat exposure.
  • Absence from work in the heat for a week or more results in a significant loss in the beneficial adaptations leading to an increase likelihood of acute dehydration, illness, or fatigue.
  • Can be regained in 2 to 3 days upon return to a hot job.
  • Appears to be better maintained by those who are physically fit.
  • Seasonal shifts in temperatures may result in difficulties.
  • Working in hot, humid environments provides adaptive benefits that also apply in hot, desert environments, and vice versa.
  • Air conditioning will not affect acclimatization.
Acclimatization in Workers
According to Ed Sobek of AssuredBio, cell viability with DNA analysis may become a non-issue in the future. "Treating a sample with propidium monazide will decompose free DNA, plus the DNA in dead and damaged Legionella cells, but the DNA of intact and healthy viable cells is unharmed," Sobek says. For more information, read "Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology" in Applied and Environmental Microbiology.