rest of september!

Hellooooo I haven't written in a long time - the days are very busy, it gets dark around 6pm! I'll try to give a brief summary of the past few weeks here!

Sept 15
I went on a "hippo cruise" with Laura, Nikki, and two other guys we know through CIDRZ: Kapata and Jim. The boatride was in Kafue, which is about 45 minutes from our house. On the ride we saw some interesting things such as a man on a bicycle with about 30 chickens strapped to the back, a lot of hand-made "animal crossing" signs, and a store called "Tiger Feed"(but did you know there aren't any tigers in zambia? or really in africa for that matter?). We passed many small villages as well with mud huts and homes made from only sticks. Once we got to our destination it was really nice to just sit back and enjoy the day! We went on a boatride at sunset and saw some hippos and interesting-looking birds. As we went down the Kafue river, we passed by dozen of those stick huts and families outside who were working on their land. It was a truely beautiful sight! The lab continues to be busy during the week and a student from University of Zambia has come to work with us and so I am now showing him how to run the dry blood spot tests.

Sept 22
This past weekend I helped out Aleen, a woman at my work, with her son's 3rd birthday party! It was a lot of fun and a good way to kind of get out of my element and relax with some cute kids! Barbara, from Alaska, has just started working in the lab as well and she came to help as well. All of my roomates are sick!! Some of them are dehydrated and they are having trouble keeping food in their stomachs. Actually three of the 4 ended up going to the hospital and needed an IV. They have been staying home from work and everything.

Sept 29
The sickness has spread to the other house, and everyone has decided I am probably the carrier because I haven't been struck yet.. no but in all seriousness, I'm pretty paranoid now!
I gave a talk at the weekly wednesday lab meeting today about PCR and contamination. I really enjoyed doing some research and learning more about the process and where along the process things can get contaminated. I know some of you readers are completely fluent in PCR and what it is, but I'll just give a short summary for those of you who may not know! Feel free to skip over it if you don't want to read - don't worry I won't be offended because I won't know :)
PCR, also known as the polymerase Chain reaction, is a method of amplifying, or making millions of copy of DNA. This is used in both tests i am running: the DNA HIV-1 Dry Blood Spot test and also the viral load test. The DNA dry blood spot test is a qualitative test, meaning it detects the presence of the RNA virus and the viral load test is quantitative, meaning it detects the amount of virus.
Before DNA is amplified, it must be present in solution. In order for this to be possible, we must extract the RNA virus and then transform it back into DNA. With the dry blood spot test, we start with 6 micrometers of blood, which comes from the heel of an infant. We wash these blood spots with a buffer solution to remove hemoglobin and impurities. Then an extraction solution is added in order to extract the RNA from the blood spot. In viral load testing, we start out with the blood plasma and extra the free RNA virus. This time the virus is extracted in a machine called the "Ampli-prep". The protein coating around the virus is lysed and suspended in a diluent solution. Magnetic beads attach to the RNA of the free virus and the rest of the plasma is washed away.
Although each test has a slightly different way to extract the RNA virus, once it is extracted, the "mastermix", which is a mixture of enzymes is added to each respective solution. One of the enzymes, taq polymerase is amazing in that it is able to perform reverse transcription to convert the RNA which was just extracted into DNA.
Now we are ready for PCR action! The samples, which now include the extracted RNA and mastermix solution, are taken to the thermocycler, which is a machine that varies the heat to specific temperatures in order to amplify the DNA. When the temperature heats up, double-stranded DNA is denatured, or unwound into single strands. The temperature then cools and primers (which are complimentary oligionucleotides also found in the mastermix) anneal to specific target areas on the DNA that correspond to the stretches associated with HIV. The temperature heats up and these new double stranded pieces are denatured. Primers again anneal to specific stretches forming double stranded DNA as the temperature cools. The pattern continues and as you can see, it is exponential! One cycle consists of heating and cooling, and there are about 30-some cycles in the hour and nine minutes that this machine goes for. In the end, over 10 million copies of DNA are produced and ready for detection.
In the old days before taq polymerase, there wasn't an enzyme that could survive at such high temperatures. This process was a lot more manual and involved a lot of extra additions of enzyme and closely monitoring the state of samples during PCR. People had to stay up over night because samples could simply not be left. So, as time consuming as the DNA-HIV run is (it takes about 9 hours from start to finish) we are really happy about enzymes such as taq!
Anyhow, in order to detect this HIV DNA, which is just so so small on its own, amplification is absolutely necessary! However, because millions upon millions of copies of DNA are made, if even a few foreign molecules are accidentally included in the sample, millions upon millions of copies are also made, thereby completely ruining results. When false positive results are given, a child is put on unecessary medication which leads to numerous negative side effects. When a false negative report is given to a child, the child is killed. Therefore, contamination - which can lead to false results- is a huge issue that needs to be dealt with head on. In my speech I described the effects of contamination in terms of spreading a rumor. If you tell your best friend a secret and an enemy(the "contaminator") overhears, after a few minutes, that person has told one other person. After a few more minutes, both of those people each tell someone. Then, each of those people tell someone, and 45 minutes later a couple million people know your secret.
So how do we avoid contamination? The preparation room and PCR room are separated by physical space and heavy double doors to the rooms in the lab. Also, aprons and gloves worn in each room must STAY in that respective room. Equipment such as pipettes and sample racks also stay in their respective rooms. We use "zero aerosol" pipette tips, which prevent aerosols, or airborne liquid molecules, from traveling and contaminating. We use powder-free gloves to avoid getting powder and skin cells in the samples. Pipette tips are also changed for each sample until after denaturation. (Denaturation occurs right after amplification and it is here that the amplifying comes to a complete halt)
I really enjoyed giving the speech and afterwards a few people came up to me and asked questions, which was great. Also, Ron has everyone come to the lab meeting, from the CIDRZ drivers, to the lab assistents, to the lab techs, to the cleaning crew.

Another thing about this week is, I realized I started feeling self concious about my appearance. Here in Zambia, everyone is pretty direct about things and being larger is seen as a very very good thing. Most of the women I encounter are extremely thin. Men comment on your appearance to your face and this happens on the streets, when you get into a bus, at the store, and also in the lab. It doesn't help that I am working with and constantly next to about 25 men in the lab everyday! I commented on cultural differences earlier and how Ngambo and I talked about what the definition of beauty is here and what it is in the states. She was so surprised when I told her that the people in the states all want to be tan and that they actually go to places to become darker. Also, being thin is very in style and celebrities go on these crazy diets to be a size 2. She tells me how people here actually put powder on their faces to look lighter and eat as many carbs as possible to be bigger! She says I should be happy with how I am. Then Sunday at church, there was a sermon about appearance! The topic was about outer appearance and how we are perceived by others. Pastor Walker described us as tents, and some tents are big and some are small, and some are sewn up from tearing, but the important thing is that the tent is a place for our spirit to live in. It was a much-needed and wonderful sermon that really helped me to focus on what is important, and realize again that I am adjusting to another culture. Also, I started thinking about what empty comments are. They are comments that are made that are casually thrown out perhaps by people who do not really know you for who you are, and they are usually comments that do not describe your inner self, but instead the outer self. Also, I am so lucky to be healthy! So in effect, I am trying to focus on the inner and realize there are changes you have to accept when going to a different culture. And I am choosing to let the wonderful parts of the zambian culture overwhelm me :)

This past friday we attempted to go to a place called "Le soleil", which was advertised to be this huge dance party and open bar night. However, after traveling along a dark and bumpy dirt road for a while, we became a little bit skeptical. Then we see there is a le soleil gas station, le soleil house, le soleil spiritual center, etc, and following the directions we ended up outside someone's house. The man walked out with two dogs and all I could think of was, ok this is when he pulls out an axe and kills us all. luckily he just asked what we were doing and our answer was "leaving" haha. So we go back to the spiritual center and call the woman in charge of the whole thing. The place is dead quiet and no one is in sight. She keeps telling us follow the music, there is music and we say this place is dead quiet. Then we are like where are you hearing the music? We are lost. Turns out she was at her home, in her bed about to go to sleep!! So we ended up all turning around and going back to Arcades, one of the main shopping centers to this bar called Times. It was really fun and I met and danced with these five zambian women until about 1:30 :) It was so fun and I didn't think about anything else except for feeling really happy, alive, and free. I also met a man named Pelvis, no joke.

So I woke up the next morning and something smelled really familiar and reminded me of college. I got up and walked around the house and then I saw it: Rain! Incredible. eventhough it gets so cloudy and overcast looking, the sky will be dark grey, it never rains here till rainy season, and once that starts - usually late october, it last till march! So Laura and I assumed it was rainy season and jumped into action! i got out my raincoat - you know, lightweight and breathable, fits into a tiny little convenient pouch, and my umbrella, and my water proof shoes! Laura opted for her sandals, raincoat and umbrella. We were so ready! We even took a picture. We get outside and, no one looked at all different than any other day. In fact, the rain stopped within a few minutes. It was rainy season at all, just a fluke. everyone knew this except us, pretty funny!

So by the time we got all ready for the rain, and talked about random stuff on the couch we left the house nearly 2 hours after we had planned. We went on the bus and sat in traffic for a while too, so we were pretty late. We had planned to meet up with Lisa, who is a housekeeper at CIDRZ. She was going to take us fabric shopping and then measure us because she makes african clothing! The fabric stores here are overwhelming, because there are just tons and tons of brightly colored cloth everywhere and people everywhere wanting to look at and touch everything! We got some fabric and I got some extra and some fluff to make rag dolls with! ( i have a plan to make them as christmas presents for the children of the people i work with at the lab) We got back to Lisa's house and she insisted that we stay for a traditional african meal of nshima (traditional staple starch, looks like mashed potatos), rape, and roasted chicken. She lives with her extended family in 3 small 2-bedroom homes, all contained in the same plot of land. Everyone was so incredibly friendly and warm and welcoming! I was taught how to cook nshima and they were saying its one of the foods you need to learn how to stir and cook properly before marriage.

We spent the whole day with Lisa and she took us into the market right by her house, which was amazing to see. She then took us to an outdoor sitting place where we could order drinks and one by one everyone in her entire extended family found us there and joined us! Lisa's brother has a car and so he said he could take us home, and everyone who could possible squish into the car went with us! We were jam-packed in there and the rest of the family who didn't come along was just giving us hugs and kisses all over the place and standing all around the car waving until we were out of sight. I remember looking out of the window, (well my entire body was was pressed against it so that wasn't difficult) and seeing all of these people who have less than what I have and seeing them all standing there so happy. Tons of kids were playing with this bag they stuffed for a soccer ball and others were running around chasing eachother. There was music in the background and I remember thinking how in the world am I going to leave this place??

sidenoooooooooote: My nightmares have stopped!