Detection of Protozoans Using Molecular Techniques in Routine Clinical Practice
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, and the goal is that the software could be used by anyone, free of charge.
ESCMID eLibrary by author CURRENT WORK
Evaluation of a 16S/18S platform including analysis of data outputs using BIONmeta.
- Evaluation parameter: To test for parasitic DNA in faecal genomic DNAs already screened by real-time PCR assays and microscopy for intestinal parasites.
ESCMID eLibrary by author STUDENT PROJECT
No. of samples: Inclusion of 64 faecal DNAs
- Only one sample per person
- The range of parasites detected as broad as possible
- Distribution ratio of ”parasites detected” and ”parasites not detected” of about 70/30 (i.e., N = 45 vs. N = 19).
- Preferably samples from patients with intestinal symptoms following a recent history of travelling.
- Samples tested by both real-time PCR for pathogenic intestinal protozoa (R070) and microscopy for larvae, ova, and cysts (R071) ESCMID eLibrary by author RESULTS
Detected by Dectected by Validated by Detected by NGS microscopy real-time PCR NGS (FECT)
Blastocystis 14 26 13/14 samples 23 (ST1, 5; ST2, pos by 6; ST3, 10; ST4, microscopy; 3; ST6, 1)* 23/26 samples pos by real- time PCR Chilomastix mesnili 1 NA 0 0 Cyclospora cayetanensis 1 NA 1 2 Dientamoeba fragilis NA 20 8 10 Endolimax nana 1 NA 1 3 Entamoeba coli 8 NA 5 6 (ST1) Entamoeba hartmanni 1 NA 1 1 Entamoeba 3 2 (E. dispar) 2 (E. dispar) 3 (E. dispar) histolytica/dispar Giardia intestinalis 3 6 0 0 Hymenolepis nana 1 NA 0 0
ESCMID eLibrary* Two cases of mixed subtypes by author RESULTS
A total of 19 samples negative by all non-NGS methods (mic and real-time PCR) were included. Using NGS, parasite DNA was found in none of these samples
There was a total of 31 samples (48%) where 100% agreement was found between NGS and non-NGS methods (including both negative and positive samples), in the sense that all parasites found by non-NGS were found by NGS
In two instances, parasites not found by non-NGS were found by NGS, namely Endolimax in a sample positive for Blastocystis by non-NGS and Cyclospora in a sample testing positive for Giardia and Blastocystis by non-NGS
NGS found Blastocystis in 23/26 samples identified as positive by real-time PCR; the 3 samples negative by NGS had real-time PCR Ct values ranging between 33 and 40 (mean for NGS-positive samples: 26 [range, 20–31]) ESCMID eLibrary by author EVALUATION SO FAR…
Still premature, but by and large, the platform so far appears to have ample sensitivity in terms of detecting - Amoebozoa • Entamoeba spp. • Endolimax spp. • Iodamoeba spp. (?) - Blastocystis - Cyclospora
A major limitation of the platform is its lack of ability to detect parasites belonging to the group of flagellaESCMID (Giardia, Dientamoeba, Chilomastix). eLibrary by author FUTURE WORK
Optimisation of primers and overall set-up to enable the detection of flagellates
Continuing awareness of the vast genetic diversity existing within many intestinal parasites (Cryptosporidium, Entamoeba, Endolimax, Iodamoeba, Blastocystis, etc.)
- Make sure that the primers match all sequences available in GenBank
- Make sure to continue exploring the genetic diversity and documenting it by submitting data to relevant databases
ESCMID eLibrary by author THE FUTURE OF THE 16S/18S PLATFORM + BIONMETA Evaluations and expanding application range
- Water samples and other environmental samples • Recreational water
- Animal samples
- FMT – screening of donor stool
- Research into the role of gut microbiota (bacteria, fungi, parasites) in human health and disease ESCMID eLibrary by author RESULTS
Blastocystis Method No. of bacterial species on No. of bacterial species on average in Blastocystis- average in Blastocystis- positive samples negative samples
NGS 93 71 All samples Blastocystis- Blastocystis- positive negative samples samples
No. of samples 64 23 41 Sum of squared differences 47363 5320 34985
Average no. of species 79 93 71
Variance within sample population 752 242 875
SD sample population 27,42 15,55 29,57
Variance population 740 231 853 ESCMIDSD population eLibrary27,20 15,21 29,21 by author RESULTS
Group 2
Group 1
Group 3
Group Mean number of species detected Group 1 29 Group 2 84 ESCMIDGroup 3 eLibrary99 by author CONCLUDING REMARKS DNA-based methods have revolutionized the detection and differentiation of intestinal parasites in the clinical microbiology laboratory
A major goal could be comprehensive detection and differentiation of 16S and non-host/non-food 18S genes in a stool samples by PCR, NGS, and analysis by bespoke software linked to robust and reliable sequence databases
Reference sequence databases are still rudimentary and a continuous goal is to investigate and document the genetic diversity within and across intestinal protist species so as to secure robust and reliable sequence databases
ESCMID eLibrary by author THANK YOU FOR YOUR ATTENTION
Acknowledgements
ESCMID eLibrary by author