Infection Connection: PCR

First 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!

Why 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.

Paint It Black

It’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.

Tomorrow 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.

Police Action

Last night, I finished reading Strange Things Happen, the autobiography of Stewart Copeland. He’s best known as the drummer for The Police, of course (and he’s my favorite drummer).

Stewart is quite a character, though, with a life lived far beyond the typical rock star experience. His father was a spy for the CIA, so he spent several years of his childhood in Lebanon, then in England, then southern California. His musical aptitude led him to the drums, and when the band that came to be known as The Police finally coalesced, I would say that his global experiences led him and the band to some distinctly non-traditional American music. One of the reasons I loved The Police was that sort of tribal beat, along with a distinct touch of reggae.

Post-Police, Stewart has gone on to make his name mainly as a composer of musical scores for movies and TV shows. His fortune made through the Police and subsequent endeavors allowed him to indulge in an expensive passion: polo. Yes...definitely an eclectic person!

I liked some of Stewart’s projects beyond The Police. One of my favorite albums is “The Rhythmatist,” in which he chronicles his travels in Africa in search of the beat (it was also a movie). It was fun to read his remarks about the movie, because apparently they just sort of made it up as they went along. The movie itself doesn’t do much for me (although the footage of him playing his drums in a cage on the savannah, surrounded by lions, is pretty awesomely cool), but I still love listening to the soundtrack.

I also enjoyed his band Animal Logic, formed with jazz bassist Stanley Clarke. Very pleasant music, with Stewart’s unmistakable sound. I love his sound, because it’s so energetic and frenetic. It sounds to me as though he has four arms, like Shiva...or maybe even ten, like Kali! He’s got so many things going on at once that it’s fun to try to listen to all the subtleties.

As with Keith Richards’ autobiography, I was fascinated by the band dynamic within The Police. Stewart writes quite a bit about their reunion tour, and as with Keith and Mick, there seems to be much conflict within the Police rank and file. Sting is obviously the superstar; Stewart recognizes that, but chafes at being told how to play his drums. (I don’t blame him.) Stewart obviously feels much love for Sting, but can only be pushed so far. There was a happy ending, as the tour wound down to them putting their differences aside and just having fun...just enjoying the magic that they could still create together. Magic, magic, maaaagic!

Also as with Keith’s book, I loved reading about the power that music has over musicians. I mean, I love listening to music, but I’m guessing that my enjoyment is maybe one-tenth of that experienced by those who actually create and play it. I can be swept away by a song...imagine playing that song to several thousand screaming fans! Stewart also mentions what he calls the “kinetic ritual” (also a song on his Klark Kent CD), in which the energy of people grooving to the same beat is gathered and magnified. (Think about seeing video of those holy roller churches, in which people get to dancing and jittering and falling down. There is always music playing.) Being the veteran of many concerts, I have to concur. There is a special energy in being in a group of fans listening to live music...I’ve exchanged many a glance with Cousin Shane at shows over the years, where we know we’re on the same page, being moved by the music, just happy to be there and to be rocking out. It really is a special feeling.

I got a chuckle out of Stewart’s almost fetishistic worship of his post-show shower. Not surprising...his playing style is so physical, and stage lights are freakin’ hot...I bet his body temperature is raised several degrees. It was always a necessity for him to have a shower, and he mentions that often enough that it’s obvious that no one had better come between him and that shower! It was also amusing to read about his kids’ excitement at getting to hang out with bands like Incubus or the Foo Fighters...kind of forgetting that their Dad is in the Rock and Roll Hall of Fame!

A very enjoyable read. I recommend it for any fan of ‘80s music, and I think it’s a must-read for any Police fan. I’m including “Regatta de Blanc” from the album of the same name. I always had a special fondness for their instrumentals, and this one always gets me moving and singing the non-lyrics. “Ee-yay-oh! Ee-yay-oh! Ee-yay-yay-yay-yay-yeahhhhhh!” Also a nice showcase for Stewart’s drumming style. Stewart’s hair may be white rather than bleached blond now, but this is music that will never grow old.

Speaking freely

Last week, the Supreme Court confirmed the right of Westboro Baptist Church to protest at the funerals of military personnel. I’m not going to dignify them with a picture here, but their general message is that American soldiers are dying because America is giving gays more and more rights. (Personally, it looks to me like American soldiers are dying because we are sending them into war zones. But that’s just me.) We all know that this group is abhorrent, and we all condemn their activities. However, the Supreme Court made the right decision here.

I think this is yet another fundamental difference between right and left. All of my liberal friends (at least those who have voiced an opinion) feel the same way that I do. We find this group a bunch of deplorable fanatics, but we also realize that they have the right to have their little protests and voice their strange and nauseating opinions. We despise them, but we know that free speech applies to them as much as it applies to the progressive protesters in Wisconsin, Indiana, and all across the country.

Palin weighed in immediately with a tweet:

Common sense & decency absent as wacko "church" allowed hate msgs spewed@ soldiers' funerals but we can't invoke God's name in public square.

If you can weed through her own special brand of word salad, it would seem that she is against the Supreme Court’s ruling. (Hey, Palin finally found a Supreme Court ruling that she disagrees with!) First of all, she’s quite wrong about not being able to invoke God’s name in the public square. She can stand on whatever street corner she wants and “spew” her godly message. She has that right, just as the Westboro bizarros can stand on a street corner (as long as they are at least 1,000 feet away from where the service is being conducted) and conduct their protests. She was called on this, of course, with people saying that she didn’t comprehend the full scope of free speech. She backtracked a bit then:

Obviously my comment meant that when we’re told we can’t say ‘God bless you’ in graduation speeches or pray before a local football game but these wackos can invoke God’s name in their hate speech while picketing our military funerals, it shows ridiculous inconsistency. I wasn’t calling for any limit on free speech, and it’s a shame some folks tried to twist my comment in that way. I was simply pointing out the irony of an often selective interpretation of free speech rights.

Well, thank you for confirming that you don’t comprehend the full scope of free speech, lady. The reason you can’t say ‘God bless you’ in a graduation speech or pray before a local football game (at least at a public school) is because of that other little thing about separation of church and state. Remember that? The government shall promote no religion. You most definitely were calling for a limit on free speech, because you were saying that a “common sense” and “decent” verdict from the Supreme Court would have stopped this group from their protests. The Supreme Court wasn’t applying “selective interpretation” of free speech rights; that was what YOU were doing.

Believe me, I have no love for the Westboro Baptists, and that is on many levels. Their religious fanaticism, their condemnation of gays, their willingness to invade the privacy of grieving families...they truly are an ignorant and hateful bunch, and I find them disgusting. However, I also realize that they are within their rights to gather and protest. Every time they do this, they confirm their sheer loathsomeness, and they are bringing no one to their side. NO ONE. If their goal is to turn the nation back to God, and bring people to “salvation,” they are failing miserably. So they can keep up their little protests. Maybe it will provide an object lesson to everyone about extremism in the religious community. I hope that good people will do what they can to shield grieving families from the hate exhibited by these people.

Free speech is for everyone. Even a quittiot: a quittin’ idiot with a Twitter account.

Got it!

After an initial kerfuffle about a backorder on my new computer...the new notebook’s here! The new notebook’s here! Just in time, too, because my previous laptop’s screen gave up the ghost yesterday. I spent much of the afternoon and evening yesterday getting the new one all set up, and getting some essential programs installed. I still need to get a new wireless keyboard, because the one I had doesn’t work with Windows 7. It’s a shopping day tomorrow!

Before you laugh with derision at my using a wireless keyboard with a laptop, let me head you off at the pass. When on vacation, I don’t take the keyboard with me. I use the laptop keyboard. But for everyday use, I find that the edge of the computer really cuts into my wrists, so I prefer a separate, larger keyboard. I also like a wireless mouse (I’m not a big fan of touchpads), and that I WILL take on vacation!

Anyway, I’m very happy with it. It’s so much faster! If I had to restart the old one, it took a good 15 minutes to get everything up and running, even after I weeded out the startup programs. This one takes a couple of minutes. Fun! Now let’s see what all I can fill it up with!

A quick note of thanks to everyone who sent good wishes about some family health problems. It seems like there is a lot going on right now, but things are looking up for most of those involved. I guess all you can do is just take these things as they come and do your best to make the right decisions and do the right things. That doesn’t always happen, but I think most people understand that it’s a difficult thing to be under such duress, and will have the needed compassion and understanding.

Not everyone will, of course. It’s coming up on one year since my Dad’s death, and I recall quite vividly the reaction of one particular person. I’m to the point where I can laugh about it now, because it showed so well the true nature of this person. Just ugly...ugly to the core. Such a person is surely rotting from the inside out. Well, the time is inching ever closer when they will experience such loss for themselves. You know what I’ll do? I’ll keep my goddamn mouth shut, because I know how it feels. I won’t gloat, because I know how it feels. I also find it a horrid thing to take any sort of glee in anyone’s death, no matter how much I dislike the person, or a person to whom they are connected. Especially when the person lost is such a kind and decent man as my Dad. How ghoulish and awful. A subsequent “apology” was laughable. Their initial reaction showed exactly how they felt, and their true character.

Ah well. I try to put such things in perspective, as well as in the past. I don’t forget about them, though.

On a happier note, the Notre Dame Fighting Irish men’s basketball team ended the regular season today on a high note, with a win at UConn! They’ve never won there before, and it was a good, hard-earned win. Next up is the Big East tournament, followed by the NCAA tourney. I keep thinking about how much Dad would have enjoyed watching this team! We always talked about how we love a true “team,” not one in which one guy is the superstar. The Irish definitely have that this year, although Ben Hansbrough is a standout player, and could possibly win Big East player of the year. I guess during the tourney this year, I’ll be cheering for two. That’s okay. I’m a mouthy broad!

O the humanity!

Sorry I’ve been a little scarce in Blogtropolis, but my laptop is dying a painful death and I haven’t been on as much lately. I’m writing this on the computer in my secret underground lair, AKA The Basement. Actually, I don’t think the computer itself is dying, but the screen is going out...my friend Simon tells me that it sounds like the inverter is going out, and that’s not an expensive fix. I’ll get it repaired eventually and use it as a backup, but in the meantime, I should have my brand new laptop by Friday! I found a good price for a new HP at TigerDirect, and I’m looking forward to a faster computer, as well as saying goodbye to Vista!

I hope you’ll all bear with me as I get things set up. I’m trying to keep up, but not doing very well at it. I’ll be back to what passes for normal soon. Since I’m not spending as much time on the computer, I’m getting more read and I practiced the piano for a while today. I’m doing pretty good with my workouts, and while doing that, listening to some of the CDs that I’ve gotten and haven’t cracked open yet. Good stuff like the Raveonettes, the Hives, the Fratellis, Garbage...lots of great music out there, and I’m loving it! I’m still keeping up on the news, too, of course. Right now, I just don’t feel much like sullying my blog with some of the idiocy I’m seeing. At the top of the list right now is Mike Huckabee and his Aw-shucks-I’m-just-a-simple-country-boy bullshit. But then I wasn’t going to write about any of that right now. I’ll save it up for later.

I will report that I stuck to my guns (haha, so to speak!) when it came to a month without mentioning Sarah Palin! I did not write a word about her here, and on Facebook, I posted nothing. I did make an occasional comment on someone else’s post, but despite the efforts of someone who shall remain nameless (*cough* Doug *cough*), I did not take the bait, and held true to my pledge. It was actually quite refreshing. I find that I’m actually rather bored with the broad now. Yeah yeah yeah, she made a Facebook entry (rather, her assbag assistant Mansour made one). Oh look, she tweeted. yawwwwwwn She just says stupid shit that everyone pays attention to, ignoring the fact that it’s stupid and it’s shit. If she decides to run for President, I’ll have plenty to say, I’m sure. For now, I find her a non-story.

Carry on, citizens. I’ll be back at full speed soon!

Then and Now

Heading out to a Notre Dame basketball game before too long, but a couple of updates.

I appreciate all the kind comments and support after I sang my Square Peg Blues. It’s very comforting to know that there are many wonderful people out there who have felt the same way, and I am proud to call them friends. I got some very positive feedback last night that I have to share with you. My Cousin Greg found me on Facebook, after recently getting on there to share pictures with his Marine buddies from the first Gulf War. He sent me a message, and I said Cool! I’ll send you a friend request! But I offered a word of caution. I said I’m a flaming liberal and I write about flaming liberal things. There are relatives on there that I’m not friends with because I know they’d hate my posts, and if they bothered him, he could always hide them. Gee, am I gun shy, or what? Well, Greg wrote this back: “You're my cousin, and i support and respect any opinion you have.”

How awesome is that? It was exactly what I needed. Thank you, Cousin Greg.

Speaking of cousins, I have more pictures from the Godfathers concert in Chicago. Actually, it’s not from the show, it’s from our visit to the Liar’s Club afterwards, where the band met up with fans. The picture at the start of this entry is one that I treasure. It’s me and Cousin Shane from probably around 1982. I know I was in college, and I was back here for a visit, staying with Shane’s family. I’m wearing my “God Save the Queen” Sex Pistols T-shirt! We stopped at a K-Mart, I believe, and while my Aunt Bert grabbed a few things, Shane and I stopped at a photo booth and got these pictures. Well, at the Liar’s Club, they had a photo booth. I said, “You know we HAVE to do it!”

So we did. We were laughing, and as I was trying to finger-comb my hair, it started taking the pictures! Shane looks all handsome and smiling, and I’m sitting there looking all dishevelled and unprepared. It wasn’t until the next day that I noticed the art work that someone had taped up in the booth, right over my head. Which makes the second picture even funnier.

Almost 30 years after the first picture, we made another fun memory. Good times!