Modern Techniques in Diagnostic Parasitology Gaza StripProf. Dr. Adnan Al-Hindi, PhDFaculty of Health SciencesMedical Laboratory Sciences Department 4-2-2015

Outline-Direct smear microscopy-Concentration techniques-Staining-Culture-Antigen detection-Antibody detection-Molecular methods

DSM-This is the conventional method in intestinal parasites detection worldwide. It is the standard gold method, where in major cases give the accurate result.-The microscopic examination of stool is needed for the recognition and identification of intestinal parasites and useful for the observation of motile protozoan trophozoites.

DSM-Many protozoa can be easily diagnosed (Entamoeba histolytica/dispar, Giardi alamblia, Entamoeba coli and others.

-Some types cannt be detected by DSM (depend on experience) but need staining like: Cryptosporidium sp.

-It is recommended to do three tests in three consecutive days.

Photo of protozoa in DSM

Concentration techniques-In light infection we can do the concentration technique as confirmatory test.

-It increases the possibility of seeing worm eggs, larvae, and protozoan cysts.

-The purpose of concentrating stool is to increase possibility to finding ova, cyst, or larvae in samples that not be able to seen by direct microscopy

Concentration techniquesSedimentation methodModified Formal- Ether sedimentation techniqueAcid- Ether sedimentation techniqueFlotation methodSaturated Salt Solution techniqueSheathers Sugar Centrifugal Flotation techniqueZinc Sulphate Centrifugal Flotation technique

Protozoa StainingIron Haematoxylin Solution A and Solution BThe background should stain grey with the protozoa light blue, and the nuclei blue-black. -Trichrome for MicrosporidiaMicrosporidial spores are ovoid and refractile and the spore wall stains bright pinkish-red. The spores are approximately 1.5 by 0.9. The background debris and bacteria are counterstained faint green.

Trichrome for ProtozoaMay be used to stain fresh stool or cultured organisms. Malachite Green is a green counterstain used to differentiate bacteriaModified Z/N Stain Pack (Cold Kinyoun) Acid fast organisms stain red, the background and other organisms stain blue:Example: Cryptosporidium sp.

Culture of protozoan parasitesThe in vitro culture of protozoan parasites involves highly complex procedures, which are subject to many variables. These parasites have very complex life cycles and, depending on the life cycle stage, may require different culture parameters.

When using cultureOnly a minority of parasitic infections are diagnosed routinely by cultural techniques.

Drugs discovery

However, in vitro cultivation is important for many reasons, some of which include: Diagnosis, antigen and antibody production, assessment of parasite immune modulating capabilities, drug screening, improvements in chemotherapy, differentiation of clinical isolates, determination of strain differences, vaccine production, development of attenuated strains, and the continued supply of viable organisms for studying host-parasite interactions.

Zuhair DardounahEvaluating the effect of selected medicinal plant materials and their synergistic effect with Metronidazole against Entamoeba histolytica

2014

Detection of Parasite Antigens

The diagnosis of human intestinal protozoa depends on microscopic detection of the various parasite stages in feces, duodenal fluid, or small intestine biopsy specimens. Since fecal examination is very labor-intensive and requires a skilled microscopist, antigen detection tests have been developed as alternatives using direct fluorescent antibody (DFA), enzyme immunoassay (EIA), and rapid, dipstick-like tests (CDC, 2014) accessed in 30-1-2015.

Antigen detection methods can be performed quickly and do not require an experienced and skilled morphologist. Much work has been accomplished on the development of antigen detection tests, resulting in commercially available reagents for the intestinal parasitesCryptosporidiumspp.,Entamoeba histolytica,Giardia duodenalis, andTrichomonas vaginalis. In addition, antigen detection tests using blood or serum are available forPlasmodiumandWuchereria bancrofti.

Specimens for antigen detection

Fresh or preserved stool samples are the appropriate specimen for antigen detection testing with most kits, but refer to the recommended collection procedures included with each specific kit.

Situations where immunodiagnosis is important

1. In early prepatent and in chronic phases of infection when the diagnostic stage of the parasite is scanty and can be missed on direct examination.

2. When the parasite cannot be precisely located for sampling e.g. visceral larva migrans.

3. When sampling is impossible or hazardous e.g. cerebral toxoplasmosis and hydatid cyst. .4. For differentiation between true and spurious infections (e.g. fascioliasis).5. For follow up after treatment.6. In epidemiological studies where large numbers of specimens can be simultaneously tested.

Amebiasis

EIA kits are commercially available for detection of fecal antigens for the diagnosis of intestinal amebiasis. Organisms of both the pathogenicE. histolyticaand the nonpathogenicEntamoeba disparstrains are morphologically identical. These assays use monoclonal antibodies that detect the galactose-inhibitable adherence protein in the pathogenicE. histolytica. The primary drawback of these assays is the requirement for fresh. Several EIA kits for antigen detection of theE. histolytica/E. dispargroup are available in the U.S., but only the TechLab kit is specific forE. histolytic

OrganismKit nameManufacturer - distributoraType of TestbCryptosporidiumspp.Crypto CELISACellabsEIAPARA-TECT Cryptosporidium Antigen 96Medical Chemical CorporationEIAProSpecT RapidRemelEIAProSpecTRemelEIACryptosporidiumTechLabEIACryptosporidiumWampoleEIACrypto CELCellabsIFAXPect CryptoRemelRapidCryptosporidiumspp./Giardia duodenalisPARA-TECT Cryptosporidium/Giardia DFA 75Medical Chemical CorporationDFAMerifluorMeridianDFAProSpecTRemelEIACrypto/Giardia CELCellabsIFAColorPAC*Becton DickinsonRapidImmunoCard STAT!*MeridianRapidXPectRemelRapidCryptosporidiumspp./Giardia duodenalis/Entamoeba histolytica/disparTriageBioSiteRapidEntamoeba histolyticaEntamoeba CELISACellabsEIAE. histolyticaWampoleEIAE. histolytica IITechLabEIAEntamoeba histolytica/E. disparProSpecTRemelEIAGiardia duodenalisGiardia CELISACellabsEIAPARA-TECT Giardia Antigen 96Medical Chemical CorporationEIAProSpecTRemelEIAGiardia IITechLabEIAGiardiaWampoleEIAGiardiaEIAAntibodies, Inc.EIAGiardia CELCellabsIFAProSpecTRemelRapidSimple-Read GiardiaMedical Chemical CorporationRapidWuchereria bancroftiFilariasis CELISACellabsEIA

Immunodiagnosis

-Serologic methods are available in cases such as toxoplasmosis, trichinosis, echinococcosis, cycticercosis, chronic schistosamiasis, or extra-intestinal amebiasis, where the organism is not readily demonstrated.

Serology (ELISA)-Copro-antigen ELISAs are commonly used to diagnose canine infection.

Molecular diagnosis parasites Microscopic examination is still considered the "gold standard" for the diagnosis of parasitic diseases. If an unequivocal identification of the parasite can not be made, the stool specimen can be analyzed using molecular techniques such as polymerase chain reaction (PCR). PCR amplified fragments can be analyzed by using restriction fragment length polymorphisms (RFLP) or DNA sequencing if further characterization is needed.

Why we use molecular diagnosis for parasites Offer greater sensitivity and specificity over the existing diagnostic tests.Differentiation between similar morphological types.3. They permit the detection of infections from very low parasitized samples including those from asymptomatic patients samples.

4. multiplexed PCR allows for the detection of multiple sequences in the same reaction tube proving useful in the diagnosis of several parasiticinfections simultaneously.

Polymerase Chain Reaction (PCR)

The PCR makes it possible to perform selective amplification from complex genomes. This technique is based on the process of denaturing a double-stranded genomic DNA template using heat. Next, the temperature is lowered to ensure that primers can anneal to their complementary sequences into the template. Thus, the elongated DNA template follows in both directions from the primer site by means of enzymatic catalysis with a thermostable DNA polymerase, generating double-stranded products .

Real-Time Polymerase Chain Reaction (RT-PCR).It is a system unlike conventional PCR, allow for the quantification of the original templates concentration through the use of various fluorescent chemistries, such as Sybergreen, Taqman probes, fluorescence resonance energy transfer (FRET), and Scorpion primers [7]. The concentration is measured through comparison to standard curves.

Loop-Mediated Isothermal Amplification (LAMP) Loop mediated isothermal amplification (LAMP) is a unique amplification method with extremely high specificity and sensitivity able to discriminate between a single nucleotide difference. It is characterized by the use of six different primers specifically designed to recognize eight distinct regions on a target gene, with amplification only occurring if all primers bind and form a product.

In the past, LAMP has been successfully applied for the rapid detection of both DNA and RNA viruses such as the West Nile and SARS viruses . Recently, parasitologists have adapted the LAMP approach for the detection of several parasitic diseases including the human parasites Entamoeba,Trypanosoma, Taenia, Plasmodium, and Cryptosporidium, the animal parasites Theilera, and Babesia and even to the identification of vector mosquitoes carrying Plasmodium and Dirofilaria immitis parasites.

Luminex xMAP Technology.Luminex is a bead-based xMAP technology (multianalyte profiling), a system that combines flow cytometry, fluorescent microspheres (beads), lasers and digital signal processing, and is capable of simultaneously measuring up to 100 different analytes in a single sample. It is possible to cover each set of microsphere beads by utilizing a reagent specifically designed for a particular bioassay.

Adapted to the study of parasites, the Luminex assay could identify multiple organism or different genotypes of one particular organism during the same reaction utilizing very low volume. The approach could prove useful in the study of antigenic diversity and drug resistance alleles and for the diagnosis of parasitic diseases.

Luminex was applied to the study of Cryptosporidium. C. hominis and C. parvum cannot be distinguished using antigen detection or serology assays. Only DNA-based approaches have been successful in doing so by exploiting the single nucleotide difference in the microsatellite-2 region (ML-2) of both species.

Ultimately DNA sequencing is the diagnosis tool of choice but it is costly, labour-intensive and time-consuming. In a recent study, Bandyopadhyay et al. successfully detected and distinguished C. hominis and C. parvum in 143 DNA extracts using Luminex technology by using oligonucleotide probes specific to the ML-2 regions of each species.Similarly in other research, Luminex technology wasable to detect all-blood stage parasite levels of the four human Plasmodium species (falciparum, vivax, malariae, and ovale) simultaneously.

Proteomics.Since proteins are the main catalysts, structural elements, signaling messengers, and molecularmachines of biological tissues, proteomic studies are able to provide substantial clinical relevance. Proteins can be utilized as biomarkers for tissues, cell types, developmental stages, and disease states as well as potential targets for drug discovery and interventional approaches.

Random Amplified Polymorphic DNA (RAPD)

Known as AP-PCR (arbitrarily primed PCR), RAPD has been extensively used for description of strains in epidemiological studies. The surveying of genomes of parasites is enhanced by the advantage that RAPD is a very simple, fast, and inexpensive technique that does not require either prior knowledge of the DNA sequence or DNA hybridization. (Ex Lesihmania 20 sp.)RAPD is particularly useful for studying the genetic structure of populations because it reveals polymorphisms in the noncoding regions of the genome

Restriction Fragment Length Polymorphism (RFLP)The RFLP technique is currently one of the most commonly used molecular methods for diagnosis of species and genotypes of parasites such as Toxoplasma gondii. This technique was first used to detect variations at the DNA level. This reaction is based on the digestion of the PCR products by restriction enzymes or endonucleases.

These enzymes cleave DNA into fragments of certain sizes, whose analysis on agarose or polyacrylamide gel results in different patterns of fragment sizes, enabling the identification. The RFLP technique is suitable for environmental samples because it permits the detection of multiple genotypes in the same sample. Ex. Cryptosporidum sp.RFLP technique can also be used in the differentiation of animal parasites, such as species of Theileria in sheep. A study conducted by Zaeemi et al. in Iran was able to differentiate Theileria lestoquardi, T. ovis, and T. annulata.

DNA ExtractionIt is necessary to extract DNA from the stool specimens for PCR detection. Click to view theDNA extraction protocolsrecommended for molecular diagnosis of intestinal parasites.

PCR Analysis

Molecular detection ofCyclospora cayetanensis,Entamoeba histolytica, andE. disparis performed at CDC by both conventional PCR and real-time PCR. Conventional PCR is available formicrosporidia.Amplified products detection Three microlitres of PCR-amplified DNA was detected by 2% agarose gel electrophoresis in TAE buffer (0.04 mM Tris-acetate, 0.001 mM EDTA, pH 8.0), stained in a solution of ethidium bromide (0.5 mgml-1) and visualized with a UV transilluminator.

PCR

Agarose gel Electrophoresis

Example E. histolytica/dispar

Preliminary results for Ecchinococus granulosus in dogsFig. 1 Gel electrophoresis of PCR product generated from Echinococcus ganulosus from dog

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Molecular findings in cattleFig.2 Gel electrophoresis of PCR product generated from Hydatid cyst from cattle

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The present study included 30 cattle and sheep samples. The results showed that 14/30 (46.6%) were positive for hydatid cyst using polymerase chain reaction.

Amal Al-MaqadmaEpidemiology of Trichomonas vaginalis infection among infertile women in Gaza city, Palestine

2014

dentification of two distinct species of Plasmodium responsible for ovale malariaPeople: Colin Sutherland,Mary Oguike, Spencer Polley (HTD), Peter Chiodini (HTD),Debbie Nolder(MRL), Martina Burke (MRL)A collaborative study between the UK Malaria Reference Laboratory, LSHTM, the Hospital for Tropical Diseases London and Mahidol University, Bangkok has recently demonstrated the existence of a previously unrecognised speciation between two forms of the Plasmodium parasite causing ovale malaria. We have named these two forms P. ovale curtisiorP. ovale wallikeri, after two recently deceased colleagues, Chris Curtis and David Walliker. Extending this work into the field, Mary Oguike and our collaborators have found that in some parts of Africa both these species co-exist in the same villages without inter-breeding or recombining. Future work with African and Asian collaborators is planned to further unravel the host preferences, epidemiology, population genetics and transmission biology of these newly recognised species.- See more at: http://www.lshtm.ac.uk/itd/iid/groups/sutherland-hallett_lab/diagnostics/#sthash.pi8CiGNh.dpuf

http://www.lshtm.ac.uk/itd/iid/groups/sutherland-hallett_lab/diagnostics/

3-2-2015