Rapid Testing in Microbiology Infinity and Beyond: Rapid Testing in Microbiology Presented By: Heather Wilson, MT (ASCP) Microbiology Supervisor

The Evolution of Rapid Testing in Microbiology (from culture to rapid testing) Organisms previously detected only in culture are now able to be identified with “Rapid Tests”. Bacteriology Staphylococcus aureus (SA) methicillin-resistant Staphylococcus aureus (MRSA) Escherichia coli O157:H7 (E.coli O157) Virology Influenza and Respiratory Syncytial Virus (RSV) Parasitology Malaria

Traditional Testing and TAT Bacterial Culture Requires inoculation of the specimen onto media and 18-24 hour incubation. Isolated pathogens are then manually worked up for identification based on colony morphology, Gram stain, nutritional requirements, metabolic and enzymatic capabilities. Gram stain of primary specimen provides rapid presumptive identification of pathogens present but must be followed up with culture for identification and antibiotic susceptibilities.

Use of selective and differential media Selective medium types support the growth of one group of organisms, but inhibit the growth of another; such as antimicrobials or dyes. Differential medium types - distinguish microorganisms based on biochemical reactions; such as fermenting lactose or enzymatic reactions resulting in color change. MRSA ChromAgar

Organism Identification and Antibiotic Susceptibility Testing (AST) API test strips manual biochemical tests requiring an additional 24 hour incubation. BioMerieux VITEK® 2 is an Automated bacterial identification and antibiotic susceptibility testing. BioMerieux VITEK® MS is an automated microbial identification system that uses an innovative mass spectrometry technology — Matrix Assisted Laser Desorption Ionization Time-of-Flight, or MALDI-TOF VITEX MS API Strip VITEX 2

Viral Culture Requires inoculation of the specimen into viral cell lines. Tube cultures are incubated and maintained for up to 21 days. - examined for Cytopathic Effect (CPE) - viral ID is confirmed with indirect fluorescent antibody (IFA) staining. normal monolayer monolayers with CPE Shell vial cultures are incubated 1-5 days. - viruses are detected by indirect fluorescent antibody (IFA) staining.

Direct Immunofluorescence Assay (DFA) directly tests for the presence of an antigen in patient specimen using a fluorescent labeled antibody (TAT ~4 hours except for weekends and holidays). Requires experienced tech to read cell cultures and interpret IFA / DFA slides.

Parasitology Exam for Malaria Requires preparation of thick and thin blood smears stained with Giemsa stain (TAT ~ 4 – 6 hours except for weekends and holidays). Gametocytes & rings in a thick smear. Rings in thin blood smears Requires precise microscopy skills, experienced tech to interpret smear.

All of these traditional testing approaches : Require highly trained technologists to perform the test Are labor intensive Have a lengthy turn around times Have staffing limitations

What workflows and instrumentation make rapid testing possible? Technologist staffing in our Specimen Receiving Area is 24/7. Technologists are supported by CLAs enabling them to focus on the rapid tests. The Cepheid GeneXpert Infinity System automated real-time PCR analyzer On-demand processing The BinaxNOW Malaria Test rapid membrane enzyme immunoassay qualitative detection specific for P.falciparum The SHIGA TOXIN QUIK CHEK Test rapid membrane enzyme immunoassay qualitative detection of Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2).

The Cepheid GeneXpert Infinity System Automated Real Time PCR platform Self contained single use cartridges

Influenza A, Influenza B and RSV (bed placement) Tests performed: Influenza A, Influenza B and RSV (bed placement) MRSA PCR (used to remove patients from contact precautions) SA Nasal Complete (pre-op screening) Minimal specimen processing Automated - Sample Extraction - Purification - Detection of nucleic acid target Random access Interfaced to SunQuest – results auto file to patient medical record. Results in ~1 hour

The BinaxNOW Malaria Test Malaria is a serious sometimes fatal disease (mostly P.falciparum) - caused by a parasite - transmitted by an infected anopheles mosquito. - four malarial species capable of infecting humans P.falciparum P.vivax P.ovale P.malariae Typical symptoms - high fever, chills, headache, muscle ache, flu- like symptoms. - can lead to kidney failure. Detects antigen specific for P.falciparum (0.1% parasitemia level) - and detects common pan-malarial antigen Test is performed on EDTA blood sample - Add sample & wash reagent sample migrates - Add wash reagent incubation Easy to read results in ~30 minutes

The SHIGA TOXIN QUIK CHEK Test E.coli is a normal gut flora The strain E.coli O157 - an enteric pathogen - produces Shiga toxin - causes hemorrhagic colitis, bloody diarrheal illness - potentionally life threatening complication - Hemolytic Uremic Syndrome (HUS) Transmission - contaminated water or food - contact with infected animals or persons. Treatment - Non-specific supportive therapy, including hydration. - Antibiotics should not be used - increase the risk of HUS. Test is performed on direct fecal specimens - diluted stool / conjugate incubation - wash step - substrate incubation Easy to read results in ~30 minutes

The use of these rapid tests result in earlier diagnosis and treatment of patients. Questions?