Tuesday, February 10, 2015

Spittin' ELISAs: Using Saliva to Detect Parasitic Infections

Over the weekend I attended a League of Legends tournament with my wonderful husband. Despite anticipating losing a day's worth of work, I wound up hanging out with another fellow parasitophile and then I got an e-mail from my advisor with a paper that I hadn't read yet. So, there I was, reading about detecting Trypanosoma cruzi via ELISA from salivary samples of modern people (well, people from 1995 anyway...). The paper's intro cited one study I was familiar with and FIVE that I wasn't. From that point on, I knew I'd be (eagerly and enthusiastically) reading a lot more over the weekend.

So this is a post for me for you. This is me reminding myself of what I learned and sharing it with you, which I suppose has been the goal of this blog all along.  I guess it just seems more overt at the moment.

Before we jump too far ahead, let's start by talking about what ELISAs are. ELISA stands for "enzyme-linked immunosorbent assay". It's a fancy phrase, but don't let it scare you too much; the concept is fairly straightforward. Essentially, when you are infected with a parasite, the parasite produces antigens that are specific to whichever parasite is infecting you. In response, your body produces antibodies that are specifically designed to deal with the infection of that parasite. ELISAs are tests that are designed to detect either the parasite antigen or the antibody produced in response to said antigen.

Each ELISA kit is parasite-specific to some degree or another. This is really dependent on the parasite. Some kits can detect antigens/antibodies produced by/in response to a particular species, while others can only detect parasites at the genus level, so you may not know exactly which parasite you have. In the medical world, this is sort of "good enough for government work" as you can often treat infections with species belonging to the same genus in the same manner effectively. (If you have a parasite that is susceptible to a the same treatment protocols as a different parasite, it doesn't really matter what you are treating for as long as the treatment works.) As parasite ELISAs are most often used in medical and veterinary settings for rapid diagnosis, the specificity is less of an issue. However, in the case of parasitological research, we have to be careful when we use ELISAs to assess parasitism. We have to pick the kits carefully and be aware of potential cross-reactivatity.

Parasite ELISAs typically work with two types of material: blood and feces. (Wooo-hoo!) As you might imagine, intestinal parasites are typically detected from feces while non-intestinal types of parasites are found via blood serum. The problem with serum is that acquiring it is invasive (and for those of us that study archaeoparasitology, not available for use). Interestingly, your body carries antigens and produces antibodies that wind up in places other than serum...say for example, in your saliva. Really brilliant people in the late 1980s and early 1990s figured out that you could detect a number of pathogens via ELISA testing using patient saliva rather than serum. The Chagas' paper I mentioned earlier cited three studies that found viral infections, two that demonstrated parasitic infections, and one that detected bacterial infections. The two parasitic infections were chronic schistosomiasis (Garcia and colleagues, 1995) and acute toxoplasmosis (Hajeer and colleagues, 1994). Plus the paper on Chagas', of course.

It seems that that literature grows silent on this topic after the 1990s. It doesn't seem that people found evidence that these previous studies were incorrect (as far as I can tell), so I'm wondering if it just fell off of people's radar or if it just became so well-accepted that people stopped writing about it. It's hard to say one way or the other from my limited knowledge of the parasite ELISA literature, but I'm excited to have found something that leads to more. (Before Saturday, only knew about the Hajeer paper, so now I have a little bit more to build off of, which is super exciting for me!)

The Moral of the Story
The point of all this is to tell those of you who, like me, knew/know very little about molecular work and its application to the field of parasitology. Testing saliva from people and animals is a quick, non-invasive way to do preliminary tests for certain types of parasites. It's amazing how far our technology has advanced and it will be interesting to see how these advancements help us to better understand parasitism in the world around us, past, present, and future. I'm hoping to find more parasite studies looking at the application of ELISA testing that utilizes saliva as the detection source, so this is also a request to keep your eyes and ears open and let me know if you know of work being done in the area. (Thanks in advance to those of you that actually do!)

Sunday, February 1, 2015

Sifting Through the Sediments: Parasite Eggs from Night Soil Contexts

While the last thing that most people would want to get in the mail is night soil, I'm one of those people who excitedly awaits for jars of this stuff to arrive. (Because one: Such material is super cool and because two: "most people" are boring.) In my last two posts, I discussed how we look for parasites in the context of mummified tissues and desiccated feces. Today, I'm going to introduce you to another great source of literal pay-dirt for archaeoparasitologists.

Night Soil Sediments from the Past
A nightman's calling card from the 1800s.
For those who haven't been introduced to the phrase "night soil", I'm referring to dirt that has human fecal components. The term originated from the olden days when people would shovel human waste out of cesspools and outhouses during the night and later usually sell the excrement to farmers as a fertilizer. Many of these people employed as "nightmen" or "night soil men" were not allowed to work during the day and made up a portion of the lowest of low classes in most places. This was early human waste management in Europe, Africa, and Asia long before modern standards of disposal.

As you've probably already figured out, there were lots of problems with sanitation in the past. Before modern indoor plumbing, people found a variety of ways to store their steaming piles of...stuff...before nightmen could come and haul it away. Some were indoor chambers for relieving oneself. This included things like chamber pots or water closets (the early ancestor to modern flushing toilets). Others were outdoor pit latrines such as the outhouse, the privy, the dunny, the biffy, or the long-drop.

As excrement, other waste materials, and soil fill these types of features, evidence of parasites is preserved in a stratigraphic way (with the oldest layers at the bottom and the youngest layers at the top). Archaeologist excavate these types of features along with middens (trash heaps), cisterns, and other collections of human waste from sites all over the world. These sediments are collected with provenience information carefully recorded and dates associated with other things recovered from the same layer.

Analyzing Latrine Sediments
Once these sediments make their way to people like me, they are examined using a sequential analysis model (or at least they should be) to gather all of the information that can possibly come from such material. To process this type of material, first we treat sediments with hydrochloric acid to react with any microfossil-binding calcium carbonate that might be present. Next, we use distilled water to screen heavy sediments and separate macrofossils (big pieces of plants, minerals, bones, insect remains, etc.) from microfossils (parasite eggs, starch granules, pollen, etc.) using the swirl technique. The macrofossils are dried and examined via a stereoscopic microscope. The microfossils are examined via light microscopy and exposed to further processing as needed.

SEM of Lycopodium spores.
Depending on the nature of the samples, we may need to use dangerous chemicals like hydrofluoric acid (for dissolving silicates) or zinc bromide (for heavy density flotation). We are very careful when using such things as they can be harmful if precautions aren't taken to protect ourselves. For example, we wear eye protection all the time...especially when we use zinc bromide because it attacks the optic nerve if splashed in your eye and can blind you. We also have lots of special safety protocols when we work with hydrofluoric acid as it can stop your heart if enough gets on your skin and you aren't treated quickly. Rule #1: Never do it alone, just in case. I'll be using this acid later this week for some sediments from Iowa. There will be two other people in the lab...one working on mites, and one hanging around in case I have any problems.

We also treat sediments with Lycopodium spore tablets just like we treat coprolites and mummy intestines. By adding a known amount of these spores as markers, we are able to quantify our microfossils and make sense of their presence in our samples. We use a microfossil concentration formula to determine the number of microfossils per unit (weight or volume) of sediment.

Taphonomic Issues
Like other kinds of archaeological materials, we must always consider the state of preservation for our samples. The addition of chemicals to break down excrement is something to be aware of in samples from certain places and time periods. As I learned from a recent set of historic samples from Missouri, the microfossils found in cisterns are fewer in number and much more degraded than those found in privies from the same time period.

We also must consider how often the sediments were exposed to abiotic agents of change, like wind and water. A series of wetting and drying episodes can degrade things as hardy as sporopollenin-laden pine tree pollen grains over time, leaving no evidence of anything that was once there. Biotic agents, like coprophagus (poop-eating) fungi, bacteria, and animals, can also affect what we find in latrine sediments.

From Privies to Parasites
Without going into too much detail about the types of microfossils found in latrine sediments, I thought I'd let all of you parasitophiles see some of the groovy parasite eggs that have been found from these contexts.

Parasite eggs recovered
from cemetery sediments.

Unfertilized  egg of
Ascaris lumbricoides

Molecular techniques have also been used on sediments to search for evidence of parasites that don't leave visible traces of themselves. Enzyme-linked immunosorbent assays (ELISAs) have been applied to samples in order to detect a number of protozoan parasites such as Giardia sp., and Entamoeba histolytica. 

Example ELISA plate.
Yellow wells = positive samples.
The Moral of the Story
Like mummies and coprolites, sifting through latrine sediments elicits reactions of both disgust and amazement from those following your work. The data that can be gathered from such studies are immense in volume and intensity. I have conducted a number of contract archaeology jobs that involved the analysis of latrine sediments, so I can literally call these types of material "pay-dirt" from both a scientific and from an economic standpoint. It is sad that so few researchers understand the importance and potential of examining latrine sediments, but at the same time this allows those of us who do see the value in these studies to work without a lot of interference. As always, these kinds of studies provide the best means of understanding what past people were eating and what exactly were eating them.