We've Matured...

Today is our second day of having samples hopefully growing in the incubator. Yesterday there were two cases of malaria---Jenny came in and took care of the case in the morning, and I took care of one in the afternoon. There has been one case of malaria today thus far, and I've got it in the incubator. Briefly, our current protocol consists of:

1. When a child has a fever, and their thick smear is positive for parasites the doctors in the clinic text message us to let us know they have a positive diagnosis. At this point, the doctors draw blood from the child. They try and draw a total of 10mLs, 5mLs into an anticoagulant tube (purple top), and 5mLs into a tube with nothing in it (the red top). On a big side note, the doctors draw blood from children on a regular basis as well, even if they do not have a fever. For example, blood is drawn every day 14 and day 28 after treatment was begun. Also, every child gets a routine blood draw every 90 days. Thus, some patients are asymptomatic; have parasites in their blood, but do not have fever. I asked the doctors if we could get this blood as well-----the problem is, if the child does not have a fever they do not read the smear right away. Most of the time those smears are not read until the end of the day or even the next day, and thus the blood has already been taking over to be frozen. Thus, if we want to get some asymptomatic blood, we need to come in and read the slides ourselves. I am still thinking about this, if they would let me (although I have a feeling I'll just be in the way), I could just quickly blow through the routine slides looking for parasites, just before they take the blood away. We'll see-----it also may be of no need if we start getting a few more cases of malaria a day. We have been averaging 2 cases a day, but sometimes there is none, and sometimes there are 6----------having 6 a few days this week would be great (for us).

2. Anyways, after the text message from the doctors, Jenny or I (who ever is on call at that time) come running to the lab with an eppendorf tube. We use a small 5mL syringe to poke into the purple top, then turn it upside down and draw out approximately 500microliters---------definitely 500microliters if the purple top is full as it should be, but if there is only a small amount of blood we accordingly decrease the amount that we take out. We then carefully (remember several of these children are likely to HIV positive) take off then needle and transfer the blood into the eppendorf tube. Then, run back up to the lab.

3. Next, we spin the parasites down, 2 minutes at 1400rpm in our fancy new Eppendorf 5415-R microcentrifuge (it is awesome).

4. Aspirate the serum, and discard, then wash with 1mL of hopefully prewarmed RPMI wash. This whole prewarming media would be much nicer if we had a water bath, which was supposed to be ordered ages ago. As it stands, we just put aliquots of RPMI and RPMI complete in the incubator at the start of the day (hopefully).

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-----------------WOOOOO, POWER OUTAGE--------------------I'll let you know in a few minutes whether everything starts back up all right, biggest worry obviously the incubator.-------------------------Well everything looks fine, the power was out for only about a minute, but the main thing is that the incubator came back on fine, that is no blown fuses, and the sensors seem to be correct, great----the voltage regulator must be doing its job.
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5. Continuing... Next we aspirate the RPMI wash and transfer the parasites into a 10mL tissue culture flask. Add 200microliters of 50%washed blood, and then add 10mL of culture media.

6. Here I take a 300microliter aliquot out, and use 50microliters of that to make a thin smear. The rest of this, 250microliters, gets frozen so that later on we can genotype the parasites. We plan to take a small aliquot daily for genotyping, to watch and see whether the genotype changes during adaptation to culture, and whether in a multiple infection, one parasite population is selected for. For those who don't know, the genotyping protocol that the Rosenthal/Dorsey papers use is genotyping MSP2 by PCR. We, well I since Jenny is heading out soon, may do some of this genotyping here, it remains to be seen.

7. Lastly pop the 10mL TC flask containing the new isolate in the incubator and wait. As of now, we take care of the parasites as we do in SF, change the media once a day, and split when above 10% parasitemia----or so that is the plan, we've only changed media once.

RESULTS THUS FAR:

Today I did smears on our two isolates from yesterday, and they look wonderful. The parasites have MATURED (and so has the lab). High "parasitaemia" actually, the morning isolate has progressed to schizonts, the afternoon isolate has progressed to nice mid-trophs. There are lots of parasites, but I can't get an accurate count----visualizing the RBCs is hard in this microscope----it could be the microscope, I just need to get used to it, or the oil is different (I think we use type A at home, don't we?, here we have type B). Also, since we don't have EDTA or Tris, we haven't made any TE, and thus we have been mixing the giemsa in PBS-----I think I will go to the Walter Reed lab and ask if we can steal some TE (shouldn't be a problem). We'll keep you informed of the parasites progress (and ours).

Well, that is that. The next immediate objectives include: 1. getting another tank of N2. It seems that one tank is going to last only 4-6 days depending on the amount we are culturing. This is dissappointing, but I think it is because the N2 we are getting (from the only distributor in Kampala) is of poor purity. In fact we did get two tanks of N2, but, after thinking again that the incubator was busted, we figured out that one of the tanks of N2 is about 12% oxygen, which is a pretty pathetic tank of N2. Also, it takes more than we are used to, to decrease the Oxygen in the incubator----I think that also is a product of the poor N2, but may just be this incubator also. Hopefully, I can talk to Oxy-gas, the distributor, and increase the purity of the N2 somehow------or at least quality control check their tanks.
Other than that we need: 2. to get -70 freezer here and up and running (it should be here tomorrow), 3. get the UV-light bulb for the TC hood. 4. Write up manuals/protocols on how to use the new equipment and take Sam and Moses through culturing. 5. Get the Cryo-dry shipper charged with liquid N2 and find the FEDEX office to ship it to you guys back home. 6. Continue culturing, hoping we can get some long-term cultures to freeze down in order to have something in the Cryo-dry shipper.

Outside of the lab, I think Terry and Dustin get here on Wednesday this week, and then Jenny and they will head out for their Ugandan Safari (all kinds of fun stuff I'll let her tell you about). I then will stay on here until August 16, of which then I'm, yes, going to go climb Mt. Kilimanjaro, hopefully summit, and then spend a few days in Zanzibar-----I begin the trek back to SF on August 28, arriving back in SF on the 30th. See you then.

Hope all is well,

Nate

WOW

Well, after what can only be considered an extraordinary set of events, we are now up and running again. It started when Jenny and I were all happy and excited to go to work Thursday morning. It was a nice cool morning, it had just rained, and no cars were on the road---thus the air was cleaner than I had ever seen/breathed it in Kampala. There were no cars on the road because Thursday was deemed a National Holiday so that everyone could go out and vote in the national referendum about allowing/introducing political parties (turnout was a paltry 5-10%, and that is probably exaggerating, but as most Ugandans will say, this referendum is meaningless anyway, the country wide referendum is basically for show, its clear that political parties have already formed, etc.). Anyways, it was a pleasant morning and we were excited to start our first full day of just lab work (not building contractoring). BUT, the incubator had other plans. When we arrived the incubator was off, and we couldn't get it turned on. After taking off the cover, a quick glance inside indicated that we had blown a fuse on one of the circuit boards----quite dissappointing given the two hours of work I spent the night before, just to find and restart the incubator on a fancy surge protector. Evidently the surge protector did not live up to its name. Just a side note, the whole lab is supposed to have a giant Voltage Regulator, yet it has not arrived, hmmm, and I've been trying for weeks to get the electrician that has the contract for the building to put in a bunch of extra mini-voltage regulators for our expensive equipment, to no avail, hmmm, hmmm. Anyways, we thought, well a fuse is easy enough to fix, and we'll just purchase the voltage regulator ourselves. After about a 2 hour search for the proper fuse, we came back, popped them in the incubator and turned it on...Nothing. Now what, did something worse happen to the incubator than originally thought? After a bit of worry we tried some other fuses----and got it to work briefly---thus we decided that the blown fuse was the problem, but that our new fuses were just not up to par. Anyways, being a national holiday, we couldn't get an electrician there to help us out with the fuse issue, thus we waited 'till today. Today an electrician came, left, and came back with some new fuses, and well, the incubator worked like a charm, great! Thus, we asked the electrician to set up the rest of the equipment on mini-voltage regulators and ground some items, etc. He left and didn't come back for quite some time, so we went out to lunch. We came back and to our happiness both the TC hood and incubator were up and running on a fancy new voltage regulator. ALTHOUGH, upon further inspection, the incubator was dented in the back, one of the nozzles was bent, and the power plug was hanging out. Also, one of our gas tanks had a different nozzle on it. Furthermore, there were scrapes in an arc-like fashion on the wall. Well, it didn't take too long to figure out that the tank with the different nozzle had fallen over and bounced off the incubator and onto the floor. We asked around if anybody heard anything, and oh yes, they heard a big crash but couldn't find what had happened. Well, the nozzle that evidently broke off the incubator took me two days to find, and having a 150 pound steel tank land on our one and only very expensive incubator, somewhat frustrated me. Nonetheless we have a spare gas tank, and thus we hooked it up, put some new fuses in the incubator, again, and started it up---it is still working fine. The next question was, what happened to the nozzle that I searched two days for, and where had the electrician run off to----his tools were still here. After an hour or two, the electrician came back, and well----wouldn't admit to doing anything, or anything happening. I was quite flabbergasted at this. I know this tank fell over, the wall marks are there, there is a dent in the incubator-----and I know somebody put the tank upright and put a new nozzle on the tank (albeit the wrong kind). Finally, after walking the electrician through the evidence, the electrician admitted that he had accidently knocked the gas tank over. I felt like a father who found an empty bottle of licquor in the room of my child, and the child tried to deny drinking it---------wow, I wonder where that analogy came from.

Anyways, the short story of this very long blog is that everything is working again, and the electrician is scared shit-less of me. Bring on the malaria.

Hope all is well,

Nate

Good News

Well, just excited to have ample internet access so I decided to blog again. We have both fixed the intermittent internet issues and trouble shot the inordinately slow incubator problem (wasn't decreasing the oxygen level below 12.8%). As for the internet, the ISP provider (InfoCom) changed some of our permissions and now we can actual "surf" the web----yet now the wireless router that Heidi brought with her just got fried in an electrical surge, and thus wireless is gone (for now). As for the oxygen, after fiddling for a couple hours and switching tanks, we figured out that one of the nitrogen tanks is, well, about 12.8% oxygen, thereby preventing us from getting below that level of O2. Now all I have to do is move through the beaurocracy to get the tank exchanged--perhaps a whole days worth of work.

Also, it is pouring rain out, so its likely that we will have more cases of malaria than we can deal with in about 7-10 days------the mosquitos will be out tonight.

On the other hand, our first culture, of which we quasi-incubated all night seems to not have taken (to be expected)------There are a couple of rings, but mostly those beautiful black dots observed in dying cultures---we'll see...

Adios for now.

Nate

WE'RE UP AND RUNNING!

Hello all,

Well 4 weeks on the dot since we arrived, and today, we are all up and running. The incubator arrived late Monday. We spent yesterday (Tuesday) putting the finishing touches on the gas connection to the incubator and filling the incubator with water etc. Then, after a 12 hour stabilization period overnight and a restart this morning to get the CO2 sensor working, we are all set (although, it seems it is taking an inordinate amount of time to decrease the O2, hopefully nothing is wrong). Thus, everything has arrived and is "working" except for the -70 freezer (presumably next week), and the UV light bulb for the TC hood (Alissa and Peter in the Rosenthal lab are working on getting that to us). "Working" is in quotes, because many things work intermittently. Despite putting in a separate water line to the millipore machine, we are still having pressure problems. Depending on how full the water tank is, determines whether the millipore machine works. Also, the internet which finally got installed last week, is extremely finicky---for reasons we don't know yet, some websites work and some don't, and nothing is consistent. We need to contact the ISP. Oh yeah, we also don't have a water bath yet, so we are storing aliquots of media in the incubator to keep it ready.

There was one case of malaria yesterday at the clinic, and we went through the motions on how we are going to transfer the parasites up to the lab. Briefly, the clinic staff text messages us when they have a positive diagnosis and we coming running to the lab with an eppendorf tube. Then, the doctors draw blood and we use a tiny syringe to remove approximately 200-500 microliters of blood from the anticoagulant purple top vacuum tube. Then pop the needle off and transfer the blood to the eppendorf tube. Hike up the hill to the lab, spin the blood, wash it once with warm RPMI, then put it in media in a 6 well plate/petri dish/small TC flask.

Now, we're just waiting for a sick child (as most of these clinical studies go, bad for the person/child, but good for us). Thus, we'll start culturing whenever the next positive diagnosis comes.

This last weekend I went whitewater rafting down the river Nile, it was great, and then on Sunday I went with Jenny and others to the Entebbe Zoo and Lake Victoria-----we all had freshly caught and deep fried Tilapia.

Hope all is well.

Nate

Almost ready

We have been working hard in the lab this week! We have been helping genotype samples from a study that just finished, and a lot of the equipment came in and needed to be set up. We have set up the tissue culture hood and the millipore machine and are really just waiting on the incubator to arrive to get started. I think on Monday we will be up and running. The big news for today is that we have high speed wireless internet in the lab now, so we can be more diligent about posting to the blog. Many of the UCSF faculty involved in various collaborations at Makerere University were here this week, so it was really great to meet them and here about all the science going on here. Well I guess that is it for now,
Jenny

Murchison Falls

Murchison Falls
Originally uploaded by jlshock.

Okay, here is the last picture from our trip. This is Murchison Falls as seen from our boat on the Nile.

elephant

murchs138
Originally uploaded by jlshock.

Here is an elephant who was obviously happy to see us!

Hippos

murchs084
Originally uploaded by jlshock.

Here are just two of the many hippos we saw on our game drive and our boat trip. Some hippos even came up to our camp and ate grass around our tent at night.

water buffalo

water buffalo
Originally uploaded by jlshock.

crocodile

crocodile
Originally uploaded by jlshock.

We saw this guy while riding in a boat up the Nile toward Murchison Falls.

crested crane-Uganda's national bird

crested crane-Uganda's national bird
Originally uploaded by jlshock.

lions

lions
Originally uploaded by jlshock.

Here are two of the lions we saw in the game reserve. There was a couple of adults and around four 5 month old lions playing with each other.

giraffe

giraffe
Originally uploaded by jlshock.

This is a giraffe that was quite close to our car during our game drive in the game reserve.

warthog

warthog
Originally uploaded by jlshock.

Well, since we are still waiting on a few pieces of equipment here, Nate and I decided to go on a side trip to Murchison Falls. Here is a lovely warthog who hung around our tent at the Red Chili campsite.

Hello from East Africa

Well hello all. This is Nate, my first posting on this blog since the beginning. Anyways, Jenny has kept you guys up with some great pictures, we obviously have many more, and hope to post more once we get internet access at the new lab. On that note, the lab is moving along, perhaps slower than wished, but we americans are always in a rush... Despite several delays, the TC hood arrived in Uganda today, and we should have it out of customs and delivered to the lab tomorrow. The millipore machine arrived on monday, and its setup and turning quasi-potable Kampala water into beautiful PCR water at the rate of 5L/Hour (I think thats the rate). We've almost got the bureaucracy cleared up to install the internet-------the building the lab is in, is shared with Case Western U. and thus every time something gets installed, nobody knows who should pay for it. Thus it takes a while to sort out. The incubator was shipped from the US today via DHL express, so hopefully we will have that by next week. Thus, we plan to begin collecting blood from children next week (even if we don't have the new incubator, we will use Fred Kironde's for a bit). We've had many adventures thus far, Kampala is a great and interesting city. The people are terribly polite and nice.

Well I must go, my time is expiring at the internet cafe.

Hope all is well,

Nate

uganda4s007

uganda4s007
Originally uploaded by jlshock.

Here is the building the lab is in at Mulago hospital. The downstairs area has the lab and the offices for UCSF. Upstairs is a data center for Case Western.

uganda4s003

uganda4s003
Originally uploaded by jlshock.

Here is a picture of the lab that Nate and I are helping set up. It is really starting to look like a lab!

The lab is almost ready!

We have been getting everything ready to start work in the lab, and it is coming along nicely. We even have air conditioning, which is great. We will start genotyping some samples for one of the studies that is just finishing up this week, while we are waiting for the rest of the lab equipment. I will try to post some more pictures, but the connection is very slow so it is quite tedious. I took some pictures of the lab, so maybe I will try and post those next.
Jenny

kids in mulago

kids in mulago
Originally uploaded by jlshock.

These were two of the many kids in Mulago who were very excited to see us and to have their picture taken.

mulago III parish

mulago III parish
Originally uploaded by jlshock.

Here is a beauty salon in Mulago III parish. This was sort of a typical building there.

Mulago III Parish

Hi everyone,
We had a great day out in Mulago III parish. We were helping Sarah recruit for a new cohort study called home based management of fever. We met with families and asked them if they would like to join the study, and met with many of the kids who followed us around in the streets. It was an eye opening experience! Mulago III is certainly different than the other parts of Kampala we have seen. We took lots of pictures so I am posting a few.
Jenny