Tuesday, March 15, 2011

Infection Connection: PCR

DNA strandFirst of all, thanks to everyone for their kind words of support. Things are definitely rough lately, but we all get by with a little help from our friends. Thank you.

I got a book shipment today, and it’s always fun to open up that box and pull out book after book of fun stuff. (One of the places I order from is Edward R. Hamilton, a place which has overstocks and closeouts. What is really cool is that if you send in your order via snail mail, using a check, it’s a flat-rate $3.50 for shipping. In this shipment I got nine books, at an average of $5 per book. What a deal!) This time I ordered from their Science and Nature catalog, and I found some good ones about Microbiology:

  • Emerging Epidemics: The Menace of New Infections
  • The Secret Life of Germs
  • Good Germs, Bad Germs
  • Case Files: Microbiology
  • The Life and Death of Smallpox
  • Vaccine

They all look pretty good. I skimmed through the Case Files one (I always loved the case studies when doing continuing education at the lab) and I was still able to figure out most of them (or at least be in the ballpark) and answer many of the questions. The first one was a farmer who owned cows and presented with a black eschar on his arm. I said, “Anthrax!” Winnah winnah chicken dinnah! Anyway, it made me think about how much I still love Microbiology, and since I was in need of a good diversion, I decided to write an Infection Connection entry I’ve been wanting to do for a while.

Polymerase Chain Reaction (PCR) is a powerful laboratory tool developed in the past couple of decades. Dr. Kary Mullis is generally credited with the development of the technique (and won the Nobel prize in Chemistry for it in 1993), although his research built upon the work of others, as do all inventions and techniques. Shoulders of giants, and all that. I recommend Mullis’s autobiography, Dancing Naked in the Mind Field. He’s brilliant, but a real character, and has put forth some pretty bizarre and disproven ideas. He credits his usage of LSD with his realization of how PCR could work. [shrugs] Who knows? It really is an amazing development in science, and maybe it took that expanded leap of imagination to get him to that point.

Anyway, it’s a pretty amazing thing, and I think by the time I’m done with you, you’ll think so, too! (haha) The way it works is that you mix up this cocktail that includes the sample to be tested, free nucleotides (Remember ACTG, the building blocks of DNA? Adenine, Cytosine, Thymine, and Guanine. They only fit together one way: Adenine pairs with Thymine, and Cytosine pairs with Guanine.), a DNA primer that will target a specific strand in the organism for which you are testing, and Taq polymerase, an enzyme derived from the bacterium Thermus aquaticus, an organism that thrives in high temperatures. (I’m sure I’m not the only person who always thought of “Tak!” from Stephen King’s book Desperation whenever saying “Taq polymerase.”) A few other things are in there, like a buffer for stability, and some various ions that facilitate the reaction. I’ll explain in a moment why Taq polymerase is so important.

When you get your cocktail together, you place it into a thermal cycler. This is an instrument that can reach high temperatures quickly, and then cool down quickly. At the high temperature (usually around 90° Celsius), the DNA strand in the sample denatures, i.e., the hydrogen bonds in the double helix are broken, and the DNA strand zips apart. Think of running a chainsaw down the rungs of a ladder. The thermal cycler then drops down to a lower temperature, around 60°C, at which point the DNA primers form hydrogen bonds (this is called the annealing phase) with the target strand of DNA. The thermal cycler then goes up to 72°C, which is the temperature where Taq polymerase is most effective, and the enzyme causes the free nucleotides in our cocktail to go to the site where the primers are and they begin to extend the strand of DNA. If the next nucleotide in the sequence is Thymine, an Adenine nucleotide will attach to it, and so on. Are you still with me?

What is essentially happening here is that we are duplicating that original strand of DNA in our patient sample. We split the strand at the high temperature; the primers attach to the target strand at the low temperature; the primers tell the nucleotides where to go at the medium temperature, and a new strand of double helix is formed. We have made an exact copy of that original strand of DNA, all in the matter of minutes (because the thermal cycler is capable of rapid temperature fluctuations). What’s next? We do it all over again. We now have two identical strands of DNA, and we do the process again and are left with four strands; the next cycle gives us eight strands. We let these cycles run for as long as necessary, usually up to 40 cycles. At the end of 25 cycles, there are more than 33 million copies of our original DNA strand. I don’t know about you, but that STILL blows my mind!

PCRWhy is this important? There are a couple of important reasons from a microbiological perspective. One, some infections involve a very low number of organisms. And if the organisms are damaged because the patient is already on antibiotics, they may not be able to be cultured. If there are only fragments of the organism available, or very low numbers, the PCR method can still target the specific DNA strand and increase it exponentially so that there are large numbers of DNA strands which we can test for. Second, traditional culture methods can take a while, and PCR can provide much quicker results. The best example I can think of is that in the lab where I worked most recently, we did a PCR test for Group B Streptococcus, an organism that can cause life-threatening infections in newborns if the mother carries the bacteria and delivers vaginally. Culture can take up to three days; PCR can be done in a matter of a couple of hours. If a woman goes into labor and hasn’t already been tested for Group B Strep, the PCR test can mean the difference between a vaginal birth with antibiotic prophylaxis and a C-section with its attendant complications. If a baby picks up Group B Strep from a vaginal birth, it can result in meningitis or septicemia, both of which can kill. Rapid diagnosis is key, and PCR provides that.

I was going to mention why Taq polymerase is so important. Because this enzyme is so heat-tolerant (it doesn’t denature at the 90°C temperatures used in the PCR process), it allows for a closed-system procedure. In other words, you can take it through numerous cycles without “killing” it, so that you don’t have to stop after every cycle and add more polymerase. You just mix up your cocktail, pop it in the cycler, and walk away until it’s done. This allowed for a practical application in the clinical lab; companies can put together systems that are affordable for larger hospital and reference labs.

There are other molecular methods in use in labs, including DNA-RNA hybridization and ligase chain reaction. These have definite differences, but they are all based on the molecular level rather than traditional culture methods. We did one molecular procedure to identify the species of mycobacteria in a culture; those are the organisms that cause TB and other infections, but they can be notoriously slow-growers. We can now identify a case of TB in a matter of several days rather than several weeks, which is hugely important from an epidemiological standpoint.

Twenty years ago, I remember my supervisor at the lab where I worked in Indianapolis telling me, “Eventually we’ll be able to take a sample and test it directly using DNA probes.” He gave the example of testing a sputum sample for TB directly, rather than processing and culture. We had just started doing DNA probe testing (sort of the beginning of molecular testing in the clinical lab) for Chlamydia and gonorrhea. Molecular methods are both more sensitive (able to detect lower numbers of organisms) and more specific (targeting only certain organisms, resulting in fewer false positives). Paul was right. It only took two decades to get to the point where molecular testing is a part of routine diagnostic testing, and the patient is the big winner here. Better, more accurate, and faster tests can mean shorter hospital stays, quicker recovery, and less chance of passing on serious infections like TB to others.

It really is a revolutionary testing method, and I am very glad that I got to see it unfold during the course of my career.

Sunday, March 13, 2011

Paint It Black

Paint it blackIt’s been a rough few days, and there are still more to come.

My stepfather-in-law was recently diagnosed with Stage III lung cancer, and is not doing well at all. He’s not strong enough to handle either chemo or radiation, so the goal now is to make him as comfortable and as pain-free as possible. In the short-term, the plan is to pick him up at the nursing home in a couple of weeks, bring him home to enjoy a NASCAR race and a martini with his son and the rest of us, and take him back after a couple of hours. We got together Friday with his son and daughter-in-law and had an honest discussion about some very difficult subjects.

Friday was also the day that my Dad had his stroke a year ago.

Another family member (no longer family by marriage, but still held in high esteem by many of us...I won’t say who it is out of respect for their privacy) is seriously ill and in the hospital recovering from surgery. The recovery isn’t going as well as planned, and all involved are very concerned.

Today was a meet-up with my family at my Dad’s grave site. While there, I also went over to my Cousin Donny’s grave, my paternal grandparents’ grave, and an uncle and aunt’s site. After that, we all went to grab a bite to eat and happily had some laughs and discussions...of course, some of the discussions were about an unsolved murder from 1904 that my sister was researching at the museum, as well as the death and destruction in Japan and the fears of a nuclear core meltdown. Good times, eh? We then watched a slide show that my niece put together. I was dealing pretty well, but I got pretty choked up at seeing tailgating pictures from when we took Dad and Cousin Shane to a Notre Dame game. You all know that was one of the special things that Dad and I shared. We were sports buddies and that was always one of our topics whenever we talked. Unfortunately, my niece included pictures of his service at the cemetery, and that was like a punch in the gut. I didn’t want to see that...didn’t want to see the looks on all of our faces as the flag was presented to Mom.

Paint it black2Tomorrow is the anniversary of his death. I know the first year is the hardest, and I know it will continue to get better and less painful. Mom told me that one of my sisters told her that she has a hole in her heart. I understand what she was saying, but I don’t feel that way. I still miss him like crazy, but he will always be in my heart.

[Side note: I’m curious what you guys think. Do you often visit the graves of loved ones? Mom and Dad were always big on that, going around to various relatives’ graves on Memorial Day. I’m kind of not that way. I still miss the ones that have gone on and always will, but I think of them every day. Visiting their grave isn’t a necessity for me. Having said that, I’ve got an idea for something I want to put on Dad’s stone. More to come.]

Anyway, I’ve been in a very dark place for a few days. I’ve felt at times that I was surrounded by death. This is unusual for me, and I know it will pass. (There is probably a certain measure of winter fatigue setting in, too.) In the meantime, I’ve tried to handle things the best I can. I know I haven’t said the right thing all the time, and as John Mellencamp sang, if I’ve done any wrong, I hope that I’m forgiven. I know there will be more tough times ahead, but I can only continue to do what I can to help others to get through them. As for myself, I have to turn my head until the darkness goes. And it will.