Clinical test modalities for toxoplamosis

The current epidimiological estimates of latent toxoplasma gondii infections world wide are set at approximately 30%. The rate among eastern European countries is near 80%.
I have to question the accuracy of these numbers, because it calls into question the rate of pseudopostive test outcomes. There is also the question of affinity and avidity of antigen antibody complexes in these tests, which weigh heavily on qualification data.

In essence, I think the seroprevalence of toxoplasma species among epidimiologically significant study groups, may be largely inaccurate.
Furthermore, there may be other homogeneous apicomplexan species infecting humans, that go undetected because of over emphasis on toxoplasma gondii spp.
For example, patients who exhibit symptoms of so-called Lyme disease, are very good candidates for these types of parasitic infections.
I would like to discuss some alternative tests, that will encompass an expanded range of species, and maintain accurate quantitative and qualitative results. These tests will have to be cost effective for routine clinical analysis.

Circulating antigen assays are the best, efficient and cheapest method for detection of toxoplasma and related apicomplexan species. {edit: includes non-antigenic proteins and foreign DNA\RNA residues} Chinese studies, have found the rSAG1 gene, in truncated form, can be easily detected in urine of newly infected and chronically infected mice. The antigen can be manufactured through expression vectors [e. coli, yeast, baculovirus] and made available for urinalysis assays. It is designed for specificity against Toxoplasma, but covers numerous strains.

PCR testing has pitfalls, mainly due to the high sensitivity of testing procedure.
Problems such as inhibition in sample, laboratory contamination, and skill of operators\technicians are often encountered.

My personal opinion is based on the above. I say finding mitochondrial antigen markers that cover a wide group of apicomplexans should be the focus of researchers in the field. Development of affordable testing motifs should be a priority, given the rate of unexplained climbs in chronic diseases that go undiagnosed in North America, and around the world. The importance of this parasite as a disease, must come into the foray of modern medical consciousness.

Discovered novel human apicomplexan parasite

I am making a claim on this parasite, but mtDNA COI barcode will be needed to do this officially.
I have discovered the connection between my gut infection, and my prostate infection. This was essential to do, before making this announcement.
It shows the ability for the microbe to migrate in the body, and infect cells.
The macrophages of prostate pick up cysts, and cannot digest them for antigen presentation.
The cysts seem to develop into sporozoits directly as a single sporozoite. They are not direct relationship to shedding gut oocysts. The macrophages eventually end up in gut, where presumably, the cysts
become active. The genome of this parasites is very diverse, and the various stages and forms are difficult to track.
This parasite in other forms or strains could be a factor in many chronic health diseases.
One diagnostic indicator, is elevation of mast cell activity.

I have included a small clip of the sporozoite to show one form and stage of growth. There is a distinct feature of this one, a small finger-like protuberance which acts like a probe. It could also release a spore, but I am not 100% sure about that. The crystal is rhodamine 6G dye, and there is background debris. The image was taken under phase contrast at high magnification. It is the orange rod-like object in middle of frame.

Challenges of live microscopy

I have just completed the observation of prostatic fluid, incubated at 37 C for 24 hours.
The object of this task, is to find any signs of apicompexia in the sample. The sample is much like surum, in that the contents contain many immune cells of haemotopoietic lineage. As mentioned in previous post, the cells are lymphocytes, monocytes, neutrophilic granulocytes, monocytic granulocytes, and mast cells.

The cells in the sample, were almost all denatured due to extremely active proteolytic proteins, defensins etc.
Evidence of parasitic infection, was proven at this time. There was active sporozoites present, with typical morphology. They were lancet-shaped, and banana-shaped, and some were deformed. The light reflection was very high, indicating tough or hard-like membrane. They had distinct, but low motiltiy.

Back to problems with live specimen. The sporozoites died quickly, from abrupt change in environment [presumably].
Several active life forms were found in specimen, which shared similar characteristics, and have me struggling for clear explanation.
Since the whole exercise is to find co-relationship between intestinal findings and prostate findings, my personal bias conflicts with my scientific objectivity. I am expecting to see direct morphological similarities, but this is not entirely the case.
This is what I observed:
Some of the orb-like opaque objects seen previously in\on macrophages of fresh specimen, have survived the lytic processes. Now they are maturing under glass, and "budding" exactly as yeast would. Some become very large, others are small, but both types will bud. If I had not seen this with intestinal oocsts, I would automatically consider yeast as my first guess.
There are no difference between looking at these, and looking at natural air bubbles, with the exception the live ones move. There is observable movement of small object inside the orb, moving back and forth within membrane. No other organelles are observable. When the object starts to bud from membrane, it retracts and moves about the membrane causing distortions. It is almost like something in an egg, trying to find a way out.
Other more advanced growth developments show the new bud, at one third size of mother cell, connected by a membrane. This is very similar to observation of intestinal oocysts.
The sporozoits, share same light frequencies so I am suspecting, the sporozoites emerge as singular unit from these "orbs". I don't have solid proof of this yet. I have seen these forms directly in live stool samples

There is something else. Small cell-like bodies emerged everywhere. They are dark, almost black and measure about 3 um [I am using phase contrast at full mag enhanced]; they look like another reproducing cell-type. There is obvious nuclear movement within cell, and it also produces bud, and orbiting granule, attached by small filament which is retracted back into cell. It has no light reflective qualities. I must question if it is residual host stem cell activity.
The UV absorbs some of the opaque orbs, not sure what this indicates [maybe tyrosine content].
I may attempt to place some images later.

Testing antigenic reaction in mast cells

I have a condition called prostatitis. The cause is officially unknown. I have suspected the cells in prostate were infected by the same parasite as intestine, but have a hard time quantifying microscopic analysis, due to large number of cells present in prostatic fluid.
Today, I did a test to see reaction of prostate cells to parasites stored in vitro.
I have the original sample which contains oocysts, and I have a culture of the sub units which are the infective agent I call 'zoites[proven by infection of cells found in saliva].
The procedure goes as follows: stimulate prostate gland using traditional method. Collect fluid on glass slide. Use dropper and isolate small specimen to incubate in vitro for later testing. Use the dropper to prepare three slides.
In the first slide, place 'zoite containing supernatant next to prostatic fluid on same slide.
In second slide, place first sample containing oocysts next to prostatic fluid.
Third slide remains as a control.

Results:
In the first slide, the 'zoites circulated among cells without eliciting any significant granular reaction from native cells.

In second slide, the oocyst containing specimen immediately activated all cells violently.

The third slide showed neutral reaction.

I have not determined that the cells are majority mast cells, same as found in saliva & gut. The nucleus is ill defined. There are some macrophages of monocyte and neutrophil lineage in sample.
The disturbing thing is; there are lymphocytes scouting surface antigens of mast cells.
Many cells are looking ruptured, or distorted. This should be indication of granular release [I hope]. There are also cells which resemble protozoal stage of parasite, and oocysts in control. These structures are different from oocysts found in stool sample [if they are oocysts] and are same size as lymphocytes. It is very difficult to tell them apart. The spheres have high amoeboid-type movement, and protuberances which cause distortions.
Many macrophages have opaque spheres in or on cell membrane which are not part of natural granules. Parasite in other form[s] may be in specimen.
I will need an antibody marker to confirm this.

As you can see, the indications are not good. The parasite may have antigenic immunity in certain stages. The cells in prostate seem less sensitive than cells found in saliva.
The connecting factor is the presents of elevated levels of mast cells.
It could unify some theories on causes of chronic disease if these type of parasites are more common than is known. If say, toxoplasma infects mast cells, it would be difficult to detect among granules. The implications of this are quite significant, given the mediation capabilities of these cells.

More on blood sample

In a previous post, I described the examination of blood from subject having MS like symptoms. The last round of cultures display fungus of some sort. I have obtained this from plate cultures, and a broth culture under incubation at body temp. 37 C.
I had both plate and broth [controls], both are negative for fungal growth.
Not sure if this is relative, but earlier observations of slide preparation before testing, showed very small objects thought to be viral. I am now contemplating if they were indeed fungal metabolites. The product was too small to be spores.
Since the subject was positive for aggressive mycoplasma, and possible L form bacteria, I wonder if the fungal products are connected, or if it is a natural occurrence.
I will have to repeat the testing with my own blood to see if I get a similar result.

Mast cells and chronic symptomology

Mast cells originate from haemapoietic precurser cell expressing CD34. It is proposed that they evolve from monocyte lineages. They are morpologically round, about 5-10 um in diameter and have densly packed granules, often obscurring the nucleus, which is about one third the size of the cell.
Mast cells are intimately involved with host defense and wound healing, and many other things.
There are primarily two different classes of mast cells; one is distributed in connective tissue, and the other in mucus membranes.
Mast cells have a very broad range of functions, and have a very diverse role in the immune system and other physiological events.
Mass cells are directly involved in the inflammatory process.
The granules have extreme biological activity, and can mediate a wide range of physiological events when released. Degranulation is stimulated by direct stress {such as injury} or by cross linking with IgE receptors and activation of complement.
I propose, that other soluble peptides, and neurotransmitters can also act as activation factors.
Mast cells present a high affinity receptor for IgE [Fc region] called FceRI.
Important to note, that mast cells have recently been attributed as defense system against parasitic infections such as intestinal worms.
Here are some of the known characteristics of granule mediators:
Histamine, proteoglycans-- mainly heparin {anticoagulant}, serine proteases, lipid mediators-{eg. eicosanoids}, prostaglandin d2, luekotreine C4, cytokines and chemkines.
They also contain serotonin, dopamine, trypase, and chamase.
Mast cells secrete many pleiotropic {multiple effects} cytokines. This means there may still be many functions of this cell yet to be discovered.
Because of the ubiquitous distribution of mast cells throughout the connective tissues, epithelial surfaces, and close proximity to blood vessles, it make their products available to a large variety of cell types including fibroblasts {like skin making cells}, glandular epithelial cells, nerves, vascular endothelial cells, smooth muscle cells, and finally cells of the immune system.
Plus, there is a myriad of lipid mediators [important to note], proteases, proteoglygans {which can also play a role in the activity and stability of proteins and signaling molecules}, and again, cytokines and chemokines.
There is new evidence that mast cell cytokine responses are regulated by pathogens\parasites and their products. This is a very important point to remember, when considering the potential symptoms attributed to chronic infections. So, in other words, mast cells can induce cytokine activity relative to the type of antigen or pathogen.

Examining blood sample of subject

I have looked at the blood sample of a person with MS-like symptoms including occasional loss of feelings in limbs among other things. He has had MRI, other scans, and multiple tests. Nothing conclusive.
The sample was old, but sealed. I have the person do this so as the cells die, they cannot dry out. This often prompts the endobionts and stealth bacteria to emerge.
I check this out using a variety of microscopic techniques.
In this case, I have found a number of "balloon" type structures distributed amongst the cells. There were a few lymphocytes present, and one had attached to a "balloon".
This indicates to me, an antigen\receptor interaction going on, telling me the entity is dangerous, or least unwanted.
I usually assume at this point, there may be a cell-disease agent in subjects system.
Cultures are prepared with controls, to see if any further information can be determined.
I this case, there was a bacteria which grew, but had to be destroyed. I believe it must have been a contamination.
The conclusion after multiple tests; the primary bacteria is mycoplasma. This particular strain was aggressive, and pleomorphic. There were very distinct morphological characteristics which give me confidence about the conclusion.
Antibiotic infused liquid culture revealed there was complete inhibited grow of bacteria.
Tetracycline was used for this experiment.

Securing indentification of ambiguous microbes

Today I want to talk about the options I am considering for proper identification of my parasite.
This is important, because there is no doubt that this organism is important in the investigation of digestive and other problematic human health issues.
It is also advantageous for developing testing modalities.

The best test for identifying this microbe is called "COI Barcode" [cytochrome oxidase] which is a protein coding region of mitochondrial DNA {mtDNA}.
The area of the gene, the first half containing approximately 648 base pairs, is the easiest to process thus the cheapest.
Here are 3 main reasons to use COI barcode.
1) easiest to recover, including degraded sources from diverse taxa
2)readily aligned for sequence comparisons
3)effective for making distinctions between closely related species including both vertebrate and invertebrate taxa.

Six practical reasons for using this barcode:
1) Works with small traces. Barcoding can identify species from "bits and pieces" including material in foodstuffs

2) Works for all stages of life. Barcoding can identify species in all it's many forms, from eggs and seeds, through larva and seedlings to adults and flowers.

3)Unmasks look-alikes. Barcoding can distinguish among species that look the same, uncovering dangerous organisms that look the same as harmless and enabling a more accurate view of biodiversity.

4) Reduces ambiguity. A barcode provides an unambiguous digital identifying feature for quantifying species, supplementing more analog gradations of words, shapes and colors.

5) makes expertise go further. Scientists can equip themselves with barcoding to speed identification of unknown organisms and facilatate rapid recognition of new species.

6) Democratizes access. A standard library of barcodes will empower many more people to callby name, the species around them.

Experimental success!

I have managed to demonstrate the infection process of 'zoites into cells which were grown in saliva.
To re-cap; I placed saliva in glass tube to grow cells. I assume they are epithelial type cells. They tend to be designed to secrete huge amounts of antimicrobial proteins. In any event, they must represent similar cells in mucosal secretions throughout bowel.
The proximal region of the small bowel, is the assumed area of parasitic infection of host [me].
The latest observation shows complete infiltration of some cells. This seems to indicate a specificity for cell type, or some discrete characteristic of particular cells. Internal protein activity of some of these cells are indicated. The activity of infective 'zoites seem to calm once positioned within cell body.
I have posted a small clip showing total infection of cell, with active protein still observable. The larger reddish particles are the 'zoites {still don't have proper term, sorry} and the small particles showing Brownian movement are the active proteins.
The clip is shown at 600-700x under dark field conditions.

Results from experiment

I wish these microbes were easier to predict. The anticipated infection of cells was not accomplished as expected. There were a few parameters in the experiment which may have altered the outcome.
First, the chemicals in the specimen supernatant [bleach, lysol disinfectant] had affects on the saliva derived cells. The cells massively amplified production of antimicrobial proteins.
The initial observation, before incubation showed 'zoites infiltrate cells. They did this slowly, by attaching to surface of membrane, and eventually slipping to the interior portion of membrane. Then, proceeded to central part of cell.
Predictable you say?
The incubation mix, showed significant drop in number of 'zoites. The amount of protein products from cells, increased a thousand fold. Many cells were lysed, and the cells that were infected, had active granulation which was making life difficult for the 'zoites.
I conclude, that these cells are not suitable housing for the 'zoites but that cellular infection is definitly the objective of these microbes.
It also demonstrates why I have problems with dehydration, and depression of eletrolytes. The cells use up salts, and other resources for the constant generation of cells, and their chemical products.
The million dollar question is; what cells are being infiltrated, and which defense mechanism is showing ineffective action against these 'zoites?
I have included a short "clip" showing infected cell, in conflict with intruding 'zoites. This is at 600-700x mag under dark field conditions. The visual information is not the best [waiting for expensive Lumenera camera to arrive], and there are no annotations. You might have to squint a bit. The 'zoites are orangeish red, and the background movement is chemical action from cell. the activity is observed at lower
left of cell.

My apologies, the transfer of clip was not as clear as expected.

Experiment to infiltrate cells with 'zoites

If you place saliva in a test tube, cells will continue to reproduce. These cells will protect themselves against invasion from microbes through TLR's and the production of basic antimicrobial proteins. They need very little attention to conditions governing growth.
Since the parasitic 'zoites [term used until formally classified] seek out cells for housing, and cannot survive antibodies, I think these cells should provide the perfect model for examining the interaction of the cell invasion process. I will place the infective agent into small tube with cells, and incubate for 12 hours. I will report in next post, with photo's.