Friday, 23 November 2012

How to get a parasite

There are in fact lots of easy ways to obtain parasites, if that’s your goal. For instance, some people purposefully infect themselves with hookworm as a potential way to relieve autoimmune problems like asthma; you can, too, by walking around African latrines barefoot or simply ordering a packet in the mail. Yum. Alternatively, you might contract Toxoplasmosis gondii by snuggling rat-eating cats on Pacific islands. But I’m getting ahead of myself. In practice for getting old, here's a long health-related story to bore you to death.

In mid November, 2010, the Hanse Explorer held position in the lee of a tiny dot of land called Jarvis Island, just south of the Equator in the central Pacific. With my legs braced against the sides of the head to combat the incessant rocking, I put in my contacts and then stared at myself for a long moment in the mirror. One side of my neck, just below the jaw, was puffy. Had I always been lopsided, and never noticed? I poked around, and felt a lumpy thing that was larger than the lumpy thing on other side. Nothing hurt, so I resolved to worry later, slapped on a new, not very effective, anti-seasickness/insanity-inducing Scopalomine patch, and headed to the mess hall before the day’s diving began.

10 days later, we finished out our expedition to the northern Line Islands in a spectacularly crazed and ineffective fashion at Kiritimati (Christmas) Atoll. Each morning I saw that the lump was still there. When I got back to San Diego, I went to a doctor. I wanted to get any needed screening taken care of, because I was planning to stop trying not to have a baby. I was surprised that the doctor told me to just go ahead; there was nothing medically I should think about in advance. I also asked about the lump. By this time I’d developed a theory, based on my extensive knowledge of human anatomy: the 5 weeks of dry-mouth associated with continuous Scopalomine use had desiccated the salivary glands in one side of my neck. The doctor agreed that this could very well be the explanation, told me to chew some sour gum to get the saliva flowing, and to follow up in a month if it was still swollen.

The middle of nowhere. From Sandin et al. 2008
How you get around in the middle of nowhere.

      At the beginning of January, I returned to Wollongong, Australia, where I’d been living for the past 10 months. A few weeks later, I discovered I was pregnant, noted that my neck was still swollen, and went to see my local doctor. She took the lump more seriously, and immediately referred me to have an ultrasound, to see an ear-nose-throat (ENT) specialist, and to have some basic blood tests.

I strongly suggest that you do everything in your power to avoid having a nasendoscopy. In this invasive procedure, the ENT sprays a mist up your nose to numb it, which has the side effect of giving you a spectacularly foul taste on the back of your tongue. Then he sticks a bendy rod with a camera on the end up your nose and then down your throat. I guess this is his way to see thoroughly into said nose-throat region, but it is not cool at all. Even worse when he decides you probably want to see the inside of your own nose and angles a giant screen such that you cannot escape viewing gross, delicate, pink insides. In my case, this revealed that these portions of my body were normal, but didn’t inform the cause of the mysterious lump. The blood results were also uninformative.

The ultrasound was less disgusting, but still un-nerving. The ultrasound technician, not really supposed to make diagnoses, casually mentioned that the lump was not in fact associated with my salivary gland, but instead my lymph node. All I knew about lymph nodes was that they got swollen in response to an infection; such prolonged reaction to an infection I didn’t otherwise notice seemed bad. Of course I Googled this immediately and learned I might have a range of diseases that cause swollen lymph nodes, including cancer.

I had to wait several days to see the ENT again to discuss the ultrasound results. He confirmed the technician’s assessment, and referred me to have a biopsy. This seemed very bad indeed. He also wanted me to have a CT scan, so he could better image what exactly was going on in there. I preferred not to irradiate the tiny bean of a human in my belly, so asked to put this off until later, in case the biopsy was informative enough.

Later that week, I dragged Adam with me to get the biopsy. This was worse than the nasendoscopy. Even if it didn’t hurt very much, I knew that I was being stuck with a needle in a region that seemed rather possible for things to go wrong if I sneezed; that needle would also remove a core sample of my actual body inside of it. How totally disgusting. The biopsy doctor actually took three core samples at different angles through the lump. I took the rest of the day off after that.

Me being very dramatic and feeling sorry for myself after my first biopsy (note vulnerable location near various large blood vessels and things).
The next week, it was back to the ENT. The biopsy didn’t show any sign of cancer, a fantastic relief. The biopsy did indicate inflammation (how surprising!), so the ENT suggested a round of antibiotics, in case whatever infection I had could be killed off easily. He also called for more blood tests.

There are several diseases that can present with no symptoms other than swollen lymph nodes in specific parts of the body. The first set of tests looked for evidence of renal function, which would check if my kidneys were working properly; Ross River virus (for reasons unclear to me—this mosquito-borne illness causes symptoms similar to Dengue fever, which I knew well, and didn’t have); Epstein-Barr virus, which causes mononucleosis (also known as glandular fever because it causes swollen neck lymph nodes) and is associated with certain types of lymphoma cancers; cytomegalovirus; and tuberculosis.

The pathology results came back negative, but the antibiotics didn’t help. There was still no clue why the lump remained. With lots of lead shielding over my torso, I consented to a CT scan of my neck; this didn’t lead to any great insights, either, except a better image of the inflamed lymph node.

My weekly visits to the ENT, pathology lab, and imaging clinic, combined with other doctor’s visits blood tests, and ultrasounds related to the pregnancy, started to get a little exhausting. I was glad the ENT was willing to squeeze me in, seeing me before or after all his other patients (he normally had a 4 month waiting list).

More blood tests were ordered, for Cat Scratch disease and Toxoplasmosis. I thought this was a bit ridiculous; I’d lived with cats my entire life until I moved to Australia, and since then had been essentially deprived of feline interaction (I guess people tend to have big horrible dogs here instead of lovely cats). But I was willing to try anything at this point; when doctors rearrange schedules to see you, it is a bad sign.

To be safe, the ENT suggested another biopsy while we waited for those results. This time he wanted a few gigantic-gauge core samples removed, instead of the previous fine-needle biopsies.

To make matters worse, a medical student was visiting during my second biopsy, so the doctor not only proceeded to extract the samples extremely slowly for her benefit, but he also explained loudly what he was doing the entire time, pointing out how the needle was piercing various parts of my innards—viewed via ultrasound—on a giant computer screen on the wall. This time I had come without Adam; the only way I survived was squeezing the fingers of the lovely old nurse to a pulp. She recommended I stop in at the bathroom before I left, to mop up some of the blood that had run down my neck and stuck my hair into mats.

On top of all of the doctor’s visits, I was also working long hours as a postdoc, stressing myself out over not being productive enough in the lab because I was so tired from my pregnancy, and stressing about the pregnancy because I knew I wasn’t supposed to stress because stress hormones are bad for the fetus. I was also, as my friend Branwen called it, the narcoleptic pregnant lady. I would lie down on the ground and take naps in the middle of walks, in the back of the car, at lunch time. The thought of eating vegetables made me queasy—inconvenient as a vegetarian. And I was even more emotional than normal, bursting into crazy sobbing fits after hearing cheesy commercials on the radio. So layering on the worry about whether I would be diagnosed with a life-threatening illness was not helpful.
At least the unexploded WWII ordinance we came across at Palmyra Atoll had long been rendered useless by saltwater, else I'd perhaps have many more things to worry about.
 The next week I went to see the ENT after work. In the waiting room, my GP called me, since she had also just gotten the blood test results: I had a recent infection with Toxoplasmosis gondii, a protozoan parasite. While about 10% of people in the US have a Toxoplasmosis infection at some point in their lives, it is generally only a problem if a woman acquires an infection during pregnancy, when it can cause severe and sometimes fatal birth defects. I was devastated at the idea of losing the baby, and already crying when the ENT called me in. He started with the good news that the large-gauge biopsy also showed no sign of cancerous cells. Neither the ENT nor my GP knew what to do next with the parasite diagnosis, so they sent me to an infectious disease specialist a few days later.

The specialist was clearly not primarily a clinician; his bedside manner was atrocious. He explained that although it was possible that the swollen lymph node was a symptom of the Toxoplasmosis infection, it was possible that the lymph node inflammation was caused by a different infection not yet identified; in this case, the Toxoplasmosis infection could have occurred more recently, after I conceived. It was therefore important to determine when exactly the infection occurred.

Toxoplasmosis is carried by cats who eat rodents. Prior to our stops at Jarvis and Kiritimati islands, the Hanse Explorer had delivered us to Palmyra Atoll, which has a population of two cats specifically brought to the island to eat the rats overrunning the place. I had snuggled these cats, who apparently had licked their bums and then cleaned their fur, which consequently had gotten parasite eggs (oocysts) stuck on it, which I then probably accidentally ate because I didn’t wash my hands after petting the cats. Or something like that. Lovely.
My attempt at simplifying the Toxoplasmosis gondii life cycle.
I had more blood drawn to test for the avidity of IgG (immunoglobulin G) Toxoplasmosis antibodies. This is essentially a measure of how well-tuned the antibodies are to the infection: poorly-tuned antibodies with low avidity indicate a very recent infection, while well-tuned antibodies with high avidity indicate an infection occurred more than four months prior.

After more waiting, the specialist gave me the result: my IgG had high avidity. This meant I had contracted the infection before getting pregnant. This also suggested that the swollen lymph node, occurring about 10 days after we visited Palmyra Atoll, was probably related to my contracting Toxoplasmosis from the island cats; happily I probably didn’t have some other dread disease.

The problem with this timing was that there was extremely little evidence in the literature to suggest whether two months between infection and conception was enough time such that my body had fully eliminated the parasite; there was a chance that the parasite was still viable after conception, and could get through the placenta and attack the baby. Infection early in pregnancy leads to worse birth defects than later infection; my baby could be blind or have severe brain damage if he was infected.

The only way to be sure the baby was not infected was through amniocentesis, so we met with the obstetrician who performs the procedure. He explained the odds to us: probably less than 1 in 6000 that the baby was infected, though the potential consequences if it were infected were large. The chance of a miscarriage due to the amniocentesis was much higher, around 1 in 200.

We decided to trust the odds, and the fact that our early ultrasounds seemed normal. We decided instead of amniocentesis, to have frequent ultrasounds to check for any abnormalities in the jellybean’s developing brain; if any were detected we could either decide to terminate the pregnancy before 20 weeks gestation, or treat the remaining infection with strong anti-malarial drugs, which could help reduce negative effects on the fetus.

All of the ultrasounds were normal, and I slowly turned into a whale. But when the due date came and went, I started to get panicky. I needed to see that my baby could see and function; that we had made the right decision. Waiting patiently until he decided to arrive was horrible.

Our baby is now a toddler, 13 months old and starting to walk on strong little legs. As far as we can tell he is completely normal (well, actually he’s perfect, from an unbiased perspective). Now I tell everyone I know who owns or tends to snuggle with random cats and might one day have a baby: insist on getting a Toxoplasmosis antibody test before you get pregnant. If you’ve already been exposed, and have developed kick-ass antibodies, you can rest easy. If not, you need to step up your accidental-cat-poop-ingestion-avoidance techniques.

Tuesday, 6 November 2012

How to lose your eyesight

I’d love to be writing up my latest research for publication right now, especially since it's Academic Writing Month. But that project is currently in the waiting-for-various-things-out-of-my-control stage, so I can’t progress. Instead of biting my nails to the quick and sending inappropriately desperate emails across the globe, I have shifted focus to some lab-based tasks. What are these things? Come along and I’ll show you what fun I have! Both tasks I’m working on this week involve squinting at tiny things.

Task 1: Are the annual growth bands in my coral cores really annual?
Much of what I do involves collecting core samples from large coral heads. Much like trees, corals grow larger with time and form annual bands within their skeletons that can be visualized using x-rays or CT scans. I then measure the width and density of these bands to calculate the coral growth rate over the length of each core, and this tells me essentially how healthy the coral was over that time period.

(A) collecting a core from a nice big coral (B) the top of the core (C) what (B) it looks like once cut into a slab and (D) an x-ray showing annual density banding. If the top band in (D) is 2006, you can count years backwards as you move down the core, going back in time.

But of course this whole concept is predicated on the idea that the bands I identify are formed yearly. In some corals the banding is clear and lovely and life is happy. My most recent cores are not this type. They have painfully vague banding, and while I’d like to think that my experience means I can successfully identify the bands despite their lack of clarity, I’d like to be sure. 

This coral has nice banding. I like it.

This coral has rather shitty banding, and makes me want to poke my eyes out.

So, what to do? I first started by trying to count the number of something called “dissepiments” in the images. You can picture a coral as a tall apartment building, one that is constantly under construction; the coral adds a new floor to the top of the building once a month. Only the top floor is occupied by living coral tissue, hard at work on construction—once one level is complete, the coral seals this off and moves upstairs to start work on another. This is a decent illustration, because the coral skeleton actually looks a lot like this on magnification. The “floors” of the apartment building are equivalent to the dissepiments. All this is to say that one way to verify whether annual bands are annual is to count dissepiments—if there are about 12 of them for each of the bands identified, you are probably on the right track.

This could be easy if the corals behaved. (Nothdurft et al. 2005)

But really they look more like this and it hurts to find those little things the red arrows are pointing to.   (From Barnes and Lough 1992)
Another method is to measure the chemistry of the coral skeleton. While the coral is constantly building its skeleton, the composition of the skeleton changes ever so slightly with changes in the surrounding water—whether due to seasonal fluctuations in temperature, sediment in the water from river runoff, etc. I can use this to analyze a particular aspect of the skeletal chemistry that I know changes seasonally every millimeter down the core. This way I can put an independent time-scale on the core and then compare this with the time-scale I got by picking out my bands. In this case, I’m using the ratio of strontium to calcium, which changes due to water temperature (the skeleton is mostly made of calcium carbonate—CaCO3—but other elements can substitute for Ca).

This is the idea. The black wiggly lines on the left show seasonal water temperature change (low in winter, high in summer), and conveniently the banding in the coral x-ray lines up with the wiggles! (From Bagnato et al. 2004)

Making these measurements is pretty straightforward but takes a lot of time:
(1) I cut the cores with a rock saw to produce a flat slab.
(2) I take the slabs to a medical facility and get them xrayed to reveal the particular convolutions of the coral growth direction in that sample.
(3) I further cut the coral slabs so that the maximum growth axis is exposed for sampling.
(4) Using an automated CNC milling machine and a lot of swearing, I grind a ledge into that exposed edge from which I’ll collect my samples.
(5) I clean out all of the powder from cutting the slab and milling the ledge that has accumulated in the coral’s pore spaces using an ultrasonic probe. This device blasts high-frequency waves through water such that tiny air bubbles form and explode, which helps clean the material, and destroy your hearing (I do wear earmuffs for this).
(6) The samples dry overnight and then I mount them on the CNC machine again and mill precise, tiny amounts of coral skeletal powder every 0.5 mm down the edge of my clean and beautiful skeletal ledge. Each of these bits of powder is caught on a square of waxed paper and then carefully transferred into a tiny plastic vial, labeled with the sample number. Too much coffee is not good for this step.
I get really excited when step 6 is over. Especially when I get to use pretty vials to spice up the lab-life.
(7) I acid-wash and dry a lot of larger plastic vials.
(8) I use a micro-balance (a very tiny and sensitive scale) to weigh out about 50 milligrams of coral powder from each of the 0.5 mm-increment samples into my clean vials. I attempt not to sneeze while doing this.
(9) I tire out my thumb using a pipette to add super-clean acid to each of the vials to dissolve the coral powder to the correct dilution.
(10) I gratefully hand the samples over to my colleague, who uses a machine called an Inductively-Coupled-Plasma-Atomic-Emission-Spectrometer to measure the Sr/Ca ratio in each of my dissolved samples.

Task 2: What’s up with the benthic foraminifera in my sand samples?

Foraminifera are single-celled marine organisms, and the “benthic” descriptor means they don’t live in the water column, but instead on the ocean bottom or on other substrates, like seagrass. Forams make complex shells, and in some areas these shells make of the majority of reef sands.

I’ve been collecting sand samples from my study sites and using a stain called Rose Bengal to dye all of the living foraminifera a lovely shade of pink. The dead shells remain white. This means that I can compare the living and the dead assemblages—the ratio of different types of foraminifera—to see if there has been a change over time (well, between “now” – alive, and “before” – dead).
Some of my pretty forams. This is from my recent paper with Sheila Walsh                                                                      
Benthic forams are sensitive to water quality—some types (A and B above) take over in dominance when the water is clear and low in nutrients, but this balance shifts (to critters like C-F above) if the water becomes nutrified (i.e. we dump sewage, fertilizers, etc. into it, or change the nutrient dynamics by removing the big tasty fish).

So how to quantify the assemblages? I scatter a scoop of my stained and dried sand sample onto a gridded tray, place it under my microscope, and then use a pin with a bit of surf wax stuck to the end to grab individual foram shells out of the sand and stick these onto little slides. Once I have enough, I count them. Voila! 

Unfortunately for my eyes, it takes a very long time to get enough—several hours per sample. And for science’s sake, I have a lot of samples…so…back to it!