DETECTION OF PROTOZOANS USING MOLECULAR TECHNIQUES IN ROUTINE CLINICAL PRACTICE C Rune Stensvold Department of Microbiology and Infection Control Statens Serum Institut Copenhagen - Denmark ECCMID 2016 in Amsterdam EW003 - Diagnosis of intestinal protozoan infections: the best way to go 2-hour Educational Workshop ESCMIDSaturday, 9 April 2016eLibrary - 08:45 - 10:45 by author FOCUS POINTS What to test for When to test for it How to test for it Current ‘state-of-the-art’ at the Laboratory of Parasitology at Statens Serum Institut and looking ahead Apology in advance for including a lot of text! I was instructed to submit the presentation for handout production, and so I put priority to including clear text messages rather than cryptic bullet points and illustrations that might be misinterpreted. ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS Jaco Verweij and colleagues ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS Jaco Verweij and colleagues ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS Jaco Verweij and colleagues ESCMID eLibrary by author THE EMERGENCE OF PCR-BASED DIAGNOSIS PCR as as a supplementary tool or as a replacement for detecting parasites, including parasites in human stool samples. - Conventional PCR and Sanger sequencing - Real-time PCR (mono, duplex, multiplex) ESCMID eLibrary by author COMPARISON OF PCR AND MICROSCOPY ESCMIDStensvold & Nielsen, eLibrary J Clin Microbiol. 2012 Feb;50(2):540-1 by author COMPARISON OF PCR AND MICROSCOPY ESCMIDVerweij & Lieshout eLibrary, Parasitology. 2011 Oct;138(12):1492-8 by author TYPICAL SET-UP Automated DNA extraction ESCMIDPCR >> eLibraryMicroscopy by author SOME ADVANTAGES OF DIAGNOSTIC PCR 1. Standardisable 2. Automatable 3. Sensitive 4. PathogenicAutomatiseret/apathogenic species 5. TypingDNA (outbreaks ekstraktion) 6. Eukaryotic diversity ESCMIDPCR >> eLibraryMicroscopy by author PCR – ISSUES AND LIMITATIONS Diagnostic PCR may not be the solution to all diagnostic issues - ”Is PCR too sensitive?” • Maybe. But no contamination issues (as opposed to fungi of clinical relevance, meaning that if the parasite is there, then it’s there) - Should/can Ct-value cut-offs be defined for some parasites? - What influences the diagnostic accuracy of PCR • Correct sampling (and how many samples?) • Test material (fresh, frozen, fixed, time until processing, etc.) • DNA extraction (do we get the DNA out of the parasites? Formalin?) • PCR assay (primer suitability, choice of reagents and PCR conditions) • Competing template (faecal DNA) ESCMID• When should results be confirmed by sequencing? eLibrary by author EMPTY GIARDIA CYSTS PCR + IFA + cyst DAPI + cyst PCR - IFA + cyst DAPI - cyst ESCMID eLibraryCourtesy of Dr Marianne Lebbad by author HELMINTHS VS GIARDIA IN TRAVELLERS (DK) 30,500 samples (10 y) – microscopy data - Helminth prevalence: 0.2% (60 positive samples) - Giardia prevalence: 4.0% ESCMID eLibraryUnpublished data by author SITUATION IN THE NETHERLANDS Helminth prevalence: 0.3% Algorithm: - Standard analysis includes real-time PCR for enteric protozoa - Microscopy for helminths and rare parasites only in cases of • Eosinophilia • Increased IgE levels (> 120 U/L) • Urticaria • Recent history of travel to the (sub)tropics • Cases of adoption/immigration Reduced costs and increased sample turnover. ESCMIDCoppenraet et al., Clin eLibraryMirobiol Infect. 2009 Sep;15(9):869-74. by author SITUATION IN THE NETHERLANDS Helminth prevalence: 0.3% Algorithm: - Standard analysis includes real-time PCR for enteric protozoa - Microscopy for helminths and rare parasites only in cases of • Eosinophilia • Increased IgE levels• NL(> 120 lab: U/L) Reduction of • Urticaria • Recent history of travelmicroscopyto the (sub)tropicsby 90% • Cases of adoption/immigration Reduced costs and increased sample turnover. ESCMIDCoppenraet et al., Clin eLibraryMirobiol Infect. 2009 Sep;15(9):869-74. by author R&D AT SSI Laboratory of Parasitology, Statens Serum Institut: - Reference laboratory for the detection of parasites in Denmark - We detect parasites in all sorts of clinical sample materials, including stool samples - We try and do a bit of research into • The influence of the gut fauna on - Human health and disease - Remaining microbiota ESCMID eLibrary by author CURRENT SITUATION AT SSI Laboratory of Parasitology, Statens Serum Institut offers the following DNA-based methods for detection of intestinal parasites: - Ascaris (real-time PCR) - Blastocystis (real-time PCR) - Blastocystis sp. subtype (conv PCR and Sanger sequencing) - Cryptosporidium spp. and Giardia intestinalis (duplex real-time PCR) - Cyclospora cayetanensis (real-time PCR in development) - Dientamoeba fragilis (real-time PCR) - Entamoeba histolytica and E. dispar (duplex real-time PCR) - Enterocytozoon bieneusi and Encephalitozoon spp. (duplex real-time PCR) - Strongyloides stercoralis (conv PCR; real-time PCR in development) - Taenia spp. (conv PCR and Sanger sequencing) ESCMID- Trichuris spp. (real-time PCR) eLibrary by author CURRENT SITUATION AT SSI Overall line of thinking: - We try to put as much ”on offer” as possible - We try to concentrate on developing the best possible services for parasites that are clinically relevant - We also provide diagnostics for some parasites, the clinical significance of which is subject to debate, e.g. Blastocystis and Dientamoeba fragilis, so as to enable ourselves and others to expand ESCMIDthe knowledge on these organisms eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. ESCMID eLibrary by author WHEN TO TEST FOR PARASITES?? In Denmark, protozoal infection is mainly suspected only in cases of persistent diarrhoea or diarrhoea in - Travellers - Immunocompromised patients - Outbreaks In Halland (Halmstad), Sweden, both Cryptosporidium and Giardia are now included on a routine basis in all cases of diarrhea, also in cases of diarrhoea acquired in Sweden in otherwise healthy individuals. ESCMID eLibrary by author INCIDENCE OF CRYPTOSPORIDIUM IN SWEDEN Incidence per 100,000 inhabitants ESCMIDhttp://www.folkhalsomyndigheten.se/amnesomraden/statistik-och -undersokningar/sjukdomsstatistik/cryptosporidiuminfektion/eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. Recently, and based on an RCT trial and other studies performed Dr Dennis Röser, we chose to remove D. fragilis from this panel due to lack of incentive for including it. ESCMID eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. Recently, and based on an RCT trial and other studies performed by Dr Dennis Röser, we chose to remove D. fragilis from this panel due to lack of incentive for including it. - In 6–12-year-old children, prevalence is 60%–80% (normal ”flora”) - Eradication of the parasite does not lead to symptom resolution ESCMID eLibrary by author CURRENT SITUATION AT SSI For many years we offered—and actually recommended—a panel of services including PCR for E. histolytica, E. dispar, Cryptosporidium, Giardia intestinalis, and Dientamoeba fragilis for routine screening of patients suspected of intestinal protozoal infection. Recently, and based on an RCT trial and other studies performed by Dr Dennis Röser, we chose to remove D. fragilis from this panel due to lack of incentive for including it. However, novel data from a large cohort study in Denmark suggest that D. fragilis is highly associated with abdominal pain and other non- diarrhoeal intestinal symptoms (unpublished observations). Moreover, the ESCMIDGPs keep testing for it (as a separate eLibraryanalysis). by author QUALITY ASSURANCE Local ring trial (Sweden) Dutch Foundation for Quality Assessment in Medical Laboratories (SKML) – Molecular Parasitology module (/Jaap van Hellemond) ESCMID eLibrary by author LOOKING AHEAD What we have: - DNA-based methods targeting specific parasites (i.e., high sensitivity and specificity but narrow range) - Microscopy, which enables detection of most gut parasites, but which is hampered by low sensitivity and specificity What we want: - A method with the broad range of microscopy and the high sensitivity and specificity of PCR. Solution: - Technology, including suitable primers, that enables amplification of the same gene for all eukaryotic organisms, with ILLUMINA-based sequencing of PCR products and analysis of DNA data using bespoke software (BIONmeta) and well-curated databases. ESCMID eLibrary by author BIONMETA – SHORT DESCRIPTION BIONmeta is a k-mer-based software for species annotation, which can use any of the existing relevant databases for annotation. The most commonly applied databases include RDP, Greengenes, SILVA, and UNITE; however, we also use a locally curated database developed at the SSI. We are going to publish some results from our evaluation studies soon,