SWACM MOLECULAR METHODS

Transcription

1 SWACM Lynnegarcia2@verizon.net LYNNE S. GARCIA, MS, FAAM, CLS, BLM Medical Chemical Corporation MOLECULAR METHODS 1

2 Where are we now? Over the last 10 to 15 years, many diagnostic labs now have facilities for performing molecular diagnostics. Technical advances, including realtime PCR (qpcr), have improved testing, such as contamination risk with amplified products. It is also possible to combine several relatively simple targets in a multiplex assay. In addition, the use of automated DNA/RNA isolation methods has made it possible to use nucleic acid-based detection techniques in a high-throughput format. 2

3 DNA ISOLATION Method Will isolation method for release of nucleic acid from cysts, spores, eggs, etc. be effective? Additional mechanical disruption may be required for efficient DNA isolation (Trichuris eggs, Entamoeba cysts). Inhibition Nucleic acids must be free of contaminating substances that interfere with amplification reaction (complex matrix such as fecal material) Internal inhibition control mandatory for each reaction Potential problems with formalin or SAF (contains formalin) 3

4 DNA ISOLATION from FECES Method More commercial products for DNA isolation available Protocols for automated DNA isolation systems are now available Approach Feces added to polyvinylpolypyrolidone (PVPP), heated, treated with sodium-dodecyl sulphate-proteinase K treatment DNA isolated using QIAamp Tissue Kit spin columns (Qiagen) All samples stored at -20ᵒC Verweij, JJ, Leiden Univ, The Netherlands 4

5 Examples of Potential Issues Cryptosporidium assays based on DNA-j sequence commonly used in diagnostic settings: will detect C. hominis and C. parvum ONLY If assay based on small subunit rrna ( SSU rrna) highly conserved among apicomplexa: other species detected as well (Cystoisospora, Cyclospora) Sequence data limited mainly to data in GenBank; does not always follow changing taxonomy Computer simulations must be challenged by using control DNA individual parasites, cultured or purified organisms, isolated fecal genomic DNAs. 5

6 PROTISTS (not animals, plants, fungi) GENERA IN HUMAN INTESTINE Amebae Entamoeba spp. E. histolytica, E. dispar, E. moshkovskii, E. bangladeshi Approach PCR used in routine diagnostic labs E. histolytica specific, E. histolytica/e. dispar group Verweij, JJ, Leiden Univ, The Netherlands 6

7 PROTISTS GENERA IN HUMAN INTESTINE Flagellates Giardia spp. G. lamblia (duodenalis, intestinalis, enterica) Dientamoeba fragilis Need to determine clinical significance of two known genotypes of D. fragilis Approach D. fragilis can be integrated in multiplex nucleicacid based detection methods Verweij, JJ, Leiden Univ, The Netherlands 7

8 PROTISTS GENERA IN HUMAN INTESTINE Apicomplexa (Cryptosporidium, Cyclospora, Cystoisospora, Sarcocystis) Cryptosporidium spp. (>20 species) Have to consider C. meleagridis Approach Genus-specific PCR: Small subunit ribosomal RNA (SSU rrna) gene, oocyst wall protein, DNAj gene (protein production) DNAj gene-based Taqman (Crypto/Giardia rapids) 8

9 PROTISTS GENERA IN HUMAN INTESTINE Apicomplexa (Cryptosporidium, Cyclospora, Cystoisospora, Sarcocystis) Cyclospora cayetanensis Approach Species-specific PCR: Small subunit ribosomal RNA (SSU rrna) gene primary target, use of multiplex screening (Cyclo, Cystoisospora, microsporidia) With addition of Crypto good combination 9

10 PROTISTS GENERA IN HUMAN INTESTINE Microsporidia (>160 genera, 1500 species) At least 14 known to infect humans Enterocytozoon bieneusi most common Also Encephalitozoon intestinalis Approach Diagnostic PCRs helpful, ID to genus/species PCR followed by micro array (E. bieneusi, E. cuniculi, E. hellem, E. intestinalis); good combination 10

11 PROTISTS GENERA IN HUMAN INTESTINE Stramenopile Blastocystis (hominis) spp. 9 strains/species/subtypes known Half pathogenic, half non-pathogenic Approach Three real-time PCRs published, subsequent melting curve analysis Nucleic-acid based diagnosed probably be implemented as first-line diagnostics (one trial run looks good) 11

12 SOIL-TRANSMITTED HELMINTHS Ascaris, Trichuris, Necator, Ancylostoma, Strongyloides Trichuris: Taqman Array Card Hookworm: PCR in feces, internal transcribed spacer (ITS2), high specificity/sensitivity Strongyloides: real-time PCR, not evaluated on clinical specimens; test based on 18 S ribosomal RNA gene sequence much better 12

13 FUTURE DIRECTIONS Multiplex real-time PCR offers sensitive/specific alternative to microscopy (Stark, Verweij) Applies to diarrhea-causing protozoa (still an issue: Dientamoeba fragilis, Blastocystis spp., microsporidia) In combination with bacterial/viral detection Depending on patient history/clinical findings, use of algorithms recommended Immune status of patients a factor Availability and cost of commercial products may determine use of molecular testing Verweij, jj, CR Stensvold, Clin Microbiol Rev 27:

14 14 COMMERCIAL PRODUCTS Immunoassays: Giardia, Cryptosporidium, Entamoeba histolytica/e. dispar group, E. histolytica, Microsporidia, Trichomonas vaginalis, Dientamoeba, Blastocystis Luminex (xtag Gastrointestinal Pathogen Panel; PCR + fluorescent bead-based detection (11 viral/bacterial/parasitic) (Giardia, Cryptosporidium): ~3 hr or more BD MAX Enteric Parasite Panel; multiplex real-time PCR (Giardia, Cryptosporidium, Entamoeba histolytica); ~3 hr BioFire Film Array (BioMérieux); nested PCR + melting curve (Giardia, Cryptosporidium, Cyclospora, Entamoeba histolytica): ~1 hr Luminex (2018) Verigene (Nanosphere); (parasites under development/fda) PCR + gold nanoparticle hybridization; ~2 hr (Blastocystis, Cryptosporidium, Cyclospora, Dientamoeba, Entamoeba histolytica, Giardia, Microsporidia, Strongyloides)

15 15 COMMERCIAL PRODUCTS Antigen detection automation/trichomonas (BD-Affirm, BD-Probe Tec Qx/Viper, GenProbe-APTIMA) Blood Parasites: Many Plasmodium rapid tests (Alere Binax NOW FDA approved); Leishmania, Trypanosoma, other parasites: tests not FDA approved FDA approval/validation studies always an issue; most testing is in-house and not commercially available FUTURE: Most laboratory developed tests for parasites are not commercially available or available only in specialized testing centers. It is important to use internal amplification controls to detect inhibition (blood, stool), Thorough validation is required before these are implemented for clinical testing.

16 THANKS QUESTIONS?? 16