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Diagnostic Parasitology

April 17, 2009

Luke Johnson

Index

• Current Methods of Detection
• Alternatives to O&P
• Molecular Testing
• The Molecular future of Parasite Detection
  • PCR
  • qPCR
• Costs
• References

Introduction: The observation and diagnosis of intestinal parasites has not essentially changed in the last 100 years, despite the great advances in science. This is partly because human parasitic infections are generally associated with developing countries with poor healthcare. However, in the United States there are multiple intestinal parasites, mostly protozoa, that infect a great many people every year. There are also several organisms that are harmless that look like parasites. The need to definitively identify theses organisms is critically important.

The method of the past hundred years or so to identify these parasites has been to stain a stool sample and look for them. This procedure is successful at identifying parasites anywhere from 20-45% of the time.¹ Not very good odds.

Serological tests are available for some of the major parasites. Most of these have been around for at least 10 years, but have not replaced O&P stool stains.

With the advancing science of molecular biology another option presents itself. The genomes of many of the major intestinal protozoa have been sequenced. If appropriate target sequences can be found, PCR can be performed to obtain a specific and extremely accurate identification of the parasite.

Current Methods of Detection

O&P: Ova and Parasite screens generally consist of collecting a fecal sample and staining it. There are several different staining techniques that can be applied depending on what organism is suspected. They include acid fast stain, trichrome stain and a few others. All of these staining techniques are explained in detail at the CDC's website for parasitology.

Efficacy: Less than 50% sensitive if your good, but more likely less than 30%. For example, if you worked in a reference laboratory and performed 200 O&P stains everyday and your sensitivity is 50%. 100 of your patients everyday will be misdiagnosed based on your findings, or they will have to pay for additional testing. If your lab is open everyday of the year over 36,000 people will be missed.

Alternatives to O&P

Serological Testing is a major alternative to microscopy. It is less complex and requires much less experience to read than microscopy. There are multiple serological tests for intestinal protozoa. They all detect parasite antigens. There are several different methods that have been developed including, direct fluorescent antibody (DFA), enzyme immunoassay (EIA), and rapid, dipstick-like tests¹. Below is a table of all the available serological tests obtained from the CDC's website.

Organism	Kit name	Manufacturer - distributora	Type of Testb
Cryptosporidium spp.	Crypto CELISA	Cellabs	EIA
	PARA-TECT™ Cryptosporidium Antigen 96	Medical Chemical Corporation	EIA
	ProSpecT Rapid	Remel	EIA
	ProSpecT	Remel	EIA
	Cryptosporidium	TechLab	EIA
	Cryptosporidium	Wampole	EIA
	Crypto CEL	Cellabs	IFA
	XPect Crypto	Remel	Rapid
Cryptosporidium spp./Giardia lamblia	PARA-TECT™ Cryptosporidium/Giardia DFA 75	Medical Chemical Corporation	DFA
	Merifluor	Meridian	DFA
	ProSpecT	Remel	EIA
	Crypto/Giardia CEL	Cellabs	IFA
	ColorPAC*	Becton Dickinson	Rapid
	ImmunoCard STAT!*	Meridian	Rapid
	XPect	Remel	Rapid
Cryptosporidium spp./Giardia lamblia/Entamoeba histolytica/dispar	Triage	BioSite	Rapid
Entamoeba histolytica	Entamoeba CELISA	Cellabs	EIA
	E. histolytica	Wampole	EIA
	E. histolytica II	TechLab	EIA
Entamoeba histolytica/E. dispar	ProSpecT	Remel	EIA
Giardia lamblia	Giardia CELISA	Cellabs	EIA
	PARA-TECT™ Giardia Antigen 96	Medical Chemical Corporation	EIA
	ProSpecT	Remel	EIA
	Giardia II	TechLab	EIA
	Giardia	Wampole	EIA
	GiardiaEIA	Antibodies, Inc.	EIA
	Giardia CEL	Cellabs	IFA
	ProSpecT	Remel	Rapid
	Simple-Read Giardia	Medical Chemical Corporation	Rapid

There are, however, problems with many of these serological tests. According to one study a certain kit,'showed 28% sensitivity and 100% specificity. It also showed that another kit,' did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR.' It was also stated that the ELISA kits, 'were 1,000 to 10,000 times less sensitive' when compared to PCR.¹

Molecular Testing

18 S Sequence targets:

All living cells contain ribosomes and therefore ribosomal RNA (rRNA). The 40S small ribosomal subunit of eukaryotes contain an 18 S (approximately 1900 base) rRNA. Each of these 18 S rRNA's are highly conserved and can be used for species identification based on their nucleotide sequence. This makes it an ideal candidate for identification purposes. Currently all the molecular techniques used to identify intestinal parasites are based on 18 S ribosomal sequences. Below is a list of each of the major parasites and the length of the 18 S sequence that is used to identify them.

	Cryptosporidium parvum PCR amplification for cryptosporidium parvum resulted in amplification of a target segment of genomic DNA of 435 bp2.
Cyclospora cayetanensis PCR amplification for cyclospora cayetanensis resulted in amplification of a target segment of genomic DNA of 308 bp3.
	Entamoeba histolytica Unfortunately at this time there are several different targets in use to identify E. histolytica. Currently a method called riboprinting is the method used by the CDC. However more indepth procedures are given in "Laboratory Diagnostic Techniques for Entamoeba species"4
Giardia lamblia PCR amplification for giardia lamblia resulted in amplification of a segment of genomic DNA of 183 bp.5

Current molecular techniques are available and capable of detecting intestinal parasites. The future of parasitology is in using these techniques not as a second choice but as a first line for detection. The primers exist for all the major intestinal protozoa. As all of the 4 major intestinal parasites give distinct bands why not run one single test instead of the many different staining techniques that are both time consuming, and open to interpretation. Below are two examples of PCR techniques that would be clearer and easier to perform.

PCR

qPCR
The first real-time fluorescent probes developed were 5 ' nuclease probes, which are commonly referred to by their proprietary name, TaqMan probes (Fig. 1A). A TaqMan probe is a short oligonucleotide (DNA) that contains a 5 fluorescent dye and 3 quenching dye. To generate a light signal (i.e., remove the effects of the quenching dye on the fluorescent dye), two events must occur. First, the probe must bind to a complementary strand of DNA at 60°C. Second, at this tem-perature, Taq polymerase, the same enzyme used for the PCR, must cleave the 5 end of the TaqMan probe (5 nuclease activity), separating the fluorescent dye from the quenching dye. A single TaqMan probe can be used for detection of amplified
target DNA.⁸

The obvious benefits of this is that TaqMan qPCR is specific, because it depends not only on amplification through PCR, but amplicon confirmation through probe hybridization. Either the target is their or not.

This link will bring up a video for the latest Corbett Life Science demonstration of a 100 sample qPCR insturument and their approach to fluorescence monitoring of the progress of amplification.

Rotor gene 6000

Costs
It is ultimately the cost and profit margin that makes or breaks a new test. The only thing that seems to be the issue in most peoples minds is "PCR costs too much". This is not true, at least when considering only the components and materials required. PCR is at a reference lab level can be very affordable. Here is a breakdown of how PCR compares to O&P staining in price:

Major Costs
Equipment $70,000/36,500 tests a year = $1.98 per test
Repair costs(per year) $14,000/36,500 tests a year = $0.38 per test

Costs per run
Primers \
Reagents $15.00
Liscencing Fees /
Tech time @ $20/hr $10

Rounding up to $30 it costs to run a PCR test.

It costs only $1 or $2 to do an O&P but it takes more tech time. So
$2 per patient for materials
$20 tech time

SO being generous and saying that there is no other cost involved O&P costs $22. The difference is only $8. In the lab this may sound like a lot of money, but consider that these are the costs to the lab. The average cost to the patient is between $300 and $400. So what is $8 when compared to the $370 you would still be making possibly. For this $8 the patient is given a better more reliable test.

(Thank you to Anna Watts, Dr. Malcom Shields and Dr. Kathy Spiegel for working out the numbers and finding me the data in this section.)

References

1.http://jcm.asm.org/cgi/content/abstract/46/5/1678?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&titleabstract=pcr+protozoa&searchid=1&FIRSTINDEX=20&fdate=1/1/2000&resourcetype=HWCIT
2.http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=167689&blobtype=pdf

3.Pieniazek NJ, Slemenda SB, da Silva AJ, Alfano EM, Arrowood MJ. PCR confirmation of infection with Cyclospora cayetanensis. Emerg Infect Dis 1996;2:357-9.

4.http://www.isu.edu/~shiemalc/courses/ClinicalChemistryII/lab%203/Entamoeba%20Molec%20ID.pdf
5.Weiss JB, van Keulen H, Nash TE. Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequences using the polymerase chain reaction. Mol Biochem Parasitol 1992;54:73-86

6.http://en.wikipedia.org/wiki/PCR

7.http://en.wikipedia.org/wiki/Gel_electrophoresis

8.http://www.isu.edu/~shiemalc/courses/ClinicalChemistryII/lab%203/qPCR%20in%20clinical%20lab%20science.pdf