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Institut für Parasitologie | Diagnostikzentrum Parasitologie - DZP
Trends und Herausforderungen in der parasitologischen Diagnostik
DZP: Diagnostische Aufträge
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2000 2010 2019Mikroskopie Immundiagnostik Molekulardiagnostik
• Molekulardiagnostik
• Immundiagnostik
• Mikroskopie
Goldstandard mikroskopische Diagnostik ?
Mittel CH-Labore Maximum DZP
Blutparasitologie Mik 15.5 18 17
Stuhlparasitologie Mik 33.4 38 37
Malaria Rapid ID 7.9 8 8
Parasitenserologie ID 28.2 30 30
Toxoplasma IgG ID 36 36 36
Toxoplasma IgM ID 16 16 16
Externe Qualitätskontrolle: UK NEQAS (November 2019)
Seegene GI-Parasite-Panel: Evaluation (DZP)
PCR oder Mik-positivn=98
Mikroskopie* vs. GI-PPn=122
Cryptosporidium# 17/17 7 12
E. histolytica@ 11/11 0 2
E. dispar@ 0/27 17 0
Giardia 9/9 4 9
Dientamoeba 5/5 7 10
Blastocystis 28/29 25 41
Cyclospora nd 0 0
Apathogene Protozoen nd 16 0
Cystoisospora 0/1 1 n/a
* 3xSAF: 25%, 2xSAF: 65%, 1xSAF:10%; # 1xZN; @ Mik: E. histolytica-Typ, danach Differenzierung durch PCR
wurmCHECK
Preis ab 01.01.2020 CHF 74.90
• DNA-IsolationFür den wurmCHECK-Test wurde eine spezielle Methode entwickelt, um die DNA aus den Wurmeiern freizusetzten.‘Bead beating’ ? (Vermutung Autor)
• MethodikDer wurmCHECK-Test basiert auf modernster DNA-Analytik. Multiplex-PCR ? / Kapillarelektrophorese ? (Vermutung Autor)
• Qualität:Ja, der wurmCHECK Test wurde in Anlehnung an die ISO 16140 Norm validiert und wird in unserem ISO-zertifizierten Labor strikte nach einer internen SOP durchgeführt.
Eine Studie mit einer renommierten europäischen Universität wurde initiiert mit dem Ziel, den wurmCHECK Assay und die traditionelle Kotuntersuchung mittels Flotationsverfahren miteinander zu vergleichen. Die Daten aus dieser Studie werden zu gegebener Zeit publiziert.
qPCR: Quantifizierung
• Hunde• Pro Probe 5 Subsamples
Ct 1(subsample 1)
Ct 2(subsample 2)
Ct 3(subsample 3)
Ct 4(subsample 4)
Ct 5(subsample 5)
Leishmania 28.6 30.2 28.3 35.2 30.7
Giardia 34.2 29.5 34.1 38.2 27.6
Babesia 31.2 32.6 31.5 31.9 30.9
‘Neue’ Parasiten: Blastocystis
?DZP
DZP
Systematik: Stramenopila(früher: Protozoon, Pilz)
• Gruppe von Eukaryoten mit zwei unterschiedlichen Geisseln
• Meist Einzeller mit Photosynthese• Bei manchen Gruppen (z. B. Kieselalgen) werden
die begeisselten Stadien nicht mehr gebildet
Blastocystis sp. in dogs housed in Italian rescue shelters
Dogs• 99 healthy dogs• 6 shelters (Lombardy)• 50-150 dogs, 2’000 – 35’000 sqm• 3 months – 14 years• pure and cross breeds• different sizes
Results (PCR/sequencing, 18S)• 21 positive samples (21%)• All ST3• No association between age, sex, size, shelter
Blastocystis Subtypes in Humans and Animals
ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 …Unknown/
MixedNon human primates 165 117 145 8 33 55 4 27
Pigs 150 3 6 59
Cattle 24 1 3 19 31
Horse 1
Deer 1
Sheep 1 1
Dog 1 3 23
Cat 2 20
Rodents 6 4 7 1
Artiodactyla 6 7 35 1 18
Birds 3 1 10 13 1 7
Humans 882 343 1399 318 9 89 118 10 3 255
Barcoding
• Barcodes: standardized genetic locus• Mitochondrial COI gene (animals)• Chloroplast genes (plants)• rDNA ITS (fungi, alternative marker in other taxa)
• Amplify barcode region by PCR with generic primers, sequence, compare withentries in databases (GenBank, BOLD)
• Mitochondria: • Organelle with own genome (13 genes)• Multi-copy• Maternally inherited (usually clonal, extremely rarely heteroplasmy)• Higher mutation rate (as compared to nuclear genes)
• COI gene• Cytochrome oxidase subunit 1, cox1, aerobic metabolism
Beispiel: Parasit in Auge und Nase
Swiss, female, 35y, Tunesia, foreign body sensation (eye), creeping sensation (nose)
The Gadget that Makes DNA Sequencing Child’s Play: MinION
The MinION cracks open biotech to the masses the way the PC democratized computing. What will we do with this newfound power?
Targeted sequencing
Anis et al. 2018. Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples. JCM 56
• 43 pathogens (viruses, bacteria, fungi, parasites)• 198 primers (in 2 pools)• Several different primer pairs per target
• 60 clinical samples • Material: tissues, feces, milk• Ion Torrent sequencing
CAPI – Coputer Assisted Parasite Identification
Li et al. 2015: A robust and automatic method for human parasite eggrecognition in microscopic images.
Recognition rates based on shape andtexture
3 Easy Steps – Less than 5 Minutes
1. Collect fecal sample («Sample Prep Tool»)
2. Process the sample with the «Reagent Dispenser» (eggs are stained with a fluorescent marker coupled to a chitin binding protein)
3. Image the sample with the «Imaging Unit»
Egg type Sample size
Rec rate
Blood fluke 647 95.7%
Whipworm 112 96.8%
Pinworm 102 95.3%
Liver fluke 98 96.5%
Roundworm(fertilized)
110 95.7%
Roundworm(non-fertilized)
110 90.2%
MALDI-TOF MS Instruments
1) Chop off parts potentiallycontaining contaminants(blood meal, gut microbiota)
2) Mechanicalhomogenization 3) Spot on plate,
air dry, add matrix
4) Determine mass spectrum 5) Compare with referencedatabase to obtain identification
Online spectra database
MALDI-TOF mass spectrometry(Matrix-Assisted Laser Desorption/Ionization Time Of Flight)
Identification of arthrood vectors
Breathomics
• Analysis of volatile organic compounds (VOCs) associated with disease in the exhaled breath
• Gas chromatography (GC) coupled to mass spectrometry (MS) techniques
Ligand-Capped Ultrapure Metal Nanoparticle Sensors for the Detection of Cutaneous Leishmaniasis Disease in Exhaled Breath.Welearegay et al. 2018
Findings…The classification success rate of human cutaneous leishmaniasispatients achieved by our sensors test was 98.2% accuracy, 96.4% sensitivity, and 100% specificity….
Do canids infected with Dirofilaria immitis release unique volatile organic compounds in their breath?Zohdy, WAAVP 2019
Findings• Significantly more unique VOCs in infected dog breath• More phenols (known attractants) in infected dogs• Alkane hydrocarbons (known repellant compounds) were found in
non-infected dogs but were largely absent from infected dogs.
Weg von der Mikroskopie? - Alternativen für die Parasitologie
Experimentell Praxisreif Aktuell eingesetzt(Routine)
Zukunft
Mikroskopie (klassisch) ja ja ja/limitiert
PCR (Sequenzierung) ja ja ja
Panel-PCRs ja ja ja
LAMP ja limitiert limitiert
NGS/Mass sequencing ja limitiert nein ja
Bilderkennung ja nein nein ja
MALDI-TOF MS ja limitiert limitiert
Atemanalyse ja nein nein ?