PAMET Continuing Education 2016

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1 PAMET Continuing Education 2016

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3 Agent of gastroenteritis Medium/method] used for routine screening/detection in stool samples Salmonella, Shigella, MacConkey, Hektoen, Bismuth sulfite,etc. Plesiomonas Campylobacter Campylobacter agar, microaerophilic, 42 C Yersinia enterocolitica or Aeromonas Enteropathogenic E. coli Vibrio cholerae Clostridium difficile Staphylococcus aureus Rotavirus Norovirus Giardia Cryptosporidium CIN agar Sorbitol MacConkey or chromogenic agar TCBS agar Toxin assay or CCFA Mannitol salt or Baird parker medium Direct antigen detection or nucleic acid amp Nucleic acid amplification Ova & parasites examination Modified acid-fast stain or immunoassay

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8 The FilmArray is an FDA-cleared multiplex PCR system that integrates sample preparation, amplification, detection and analysis The FilmArray now has 3 FDA-cleared panels the Respiratory Panel, Blood Culture ID Panel, & the Gastrointestinal Panel.

9 The FilmArray reagent pouch stores all the necessary reagents for sample prep, reverse transcription, PCR & detection in a freeze-dried format. Sample is collected in Cary Blair transport media. Prior to a run, user injects hydration solution & sample combined with sample buffer mix into the pouch. FilmArray extracts & purifies nucleic acids from the sample. Next, it performs a nested multiplex PCR. During the first-stage PCR, the FilmArray performs a single, large volume, massively multiplexed reaction. Last, individual singleplex second-stage PCR reactions detect the products from the first stage PCR. Using endpoint melting curve data, the FilmArray software automatically generates a result for each target in a single report.

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14 Slide agglutination serotyping for H (flagellar) O (somatic) Vi (capsular) antigens

15 Foodborne outbreaks Associated with commercial produce (may be multistate) Associated with a restaurant (local outbreak) State regulations require submission of Salmonella isolates for serotyping But if Salmonella is identified by PCR, how can the serotyping be done?

16 888 people infected with the outbreak strains of Salmonella Poona have been reported from 39 states Public health officials have interviewed 38 of these ill people. Twenty-four (63%) of them reported eating cucumbers in the week before their illness started. Interviews have not identified any additional food items potentially linked with illness

17 One alternative: inoculate the stool sample into a nonselective broth medium, and send that to the public health lab along with the NAA results Burden of isolating and identifying specific pathogen falls on the public health lab Another alternative: based on the NAA result, the primary lab would inoculate the appropriate selective medium and isolate the pathogen identified by NAA Labs performing primary culture are not likely to have a broad range of selective media if they are using NAA

18 VIRUSES Isolation Nucleic acid amplif Antigen detection Adenovirus A A A Coronavirus C A C Metapneumovirus B A A Rhinovirus A A C Influenza A A A Parainfluenza A A A Resp. syncytial A A A BACTERIA Bordetella pertussis A A D Chlamyd pneumon A A B Mycoplasma pneum B A D A = useful B = sometimes useful C = seldom useful D= not available

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22 BioFire FilmArray Respiratory Panel Viruses Adenovirus Coronavirus HKU1 Coronavirus NL63 Coronavirus 229E Coronavirus OC43 Human Metapneumovirus Human Rhinovirus/ Enterovirus Influenza A Influenza A/H1 Influenza A/H Influenza A/H3 Influenza B Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Parainfluenza 4 Respiratory Syncytial Virus Bacteria Bordetella pertussis Chlamydophila pneumoniae Mycoplasma pneumoniae

23 Respiratory Viral Panel (RVP) PCR utilizing Luminex xtag Tests included in this Panel Human Metapneumovirus (hmpv) Rhinovirus Influenza A Influenza A subtype H1 Influenza A subtype H3 Influenza B Respiratory Syncytial Virus (RSV) A Respiratory Syncytial Virus (RSV) B Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Adenovirus See more at: Catalog/Detail/Respiratory-Viral-Panel-RVP- PCR#sthash.Vzt0uHOH.dpuf

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25 Using specific primers for each of the respiratory viruses, amplify nucleic acid segments in a multiplex PCR Each primer has a specific fluorescent label To look for 11 respiratory viruses, hybridize the PCR product with colored luminex beads, each color of bead hybridized with a specific NA probe After the hybridization, run the beads through a Luminex flow cytometer

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28 Diatherix TemPCR Upper Respiratory Infection Panel Laboratory in Huntsville, Alabama offers: Adenovirus types 3, 4, 7, 21 Enterovirus group Human bocavirus Human coronavirus (4 types) Human metapneumovirus Influenza A - Human influenza Influenza A - H1N1-09 Influenza B Parainfluenza types 1, 2, 3, 4 Respiratory Syncytial Virus Rhinovirus Bordetella pertussis Chlamydophila pneumoniae Haemophilus influenzae Haemophilus influenzae (Type B) Moraxella catarrhalis Mycoplasma pneumoniae Neisseria meningitidis Streptococcus pneumoniae Streptococcus pyogenes (Group A)

29 Evaluate if flu vaccine is effective against the strains prevailing in the community Help to decide on the components of the flu vaccine for the following year But traditional strain typing by hemagglutination inhibition (HAI) requires having a viral isolate, grown in cell culture

30 HAI: virus is isolated in cell culture, then identified with specific antisera

31 2 classes of drugs may be effective vs. flu A: M2 drugs amantidine/rimantadine, and neuraminidase inhibitors zanamivir and oseltamivir (Tamiflu) Nucleic acid (NA) sequencing or enzyme inhibition assays can be done on viruses isolated in cell culture But NA sequencing can also be done directly from clinical specimens by using specific DNA primers and amplifying the gene to be sequenced directly from the specimen IN THE FUTURE, both drug susceptibility testing and strain typing will likely be done by sequencing nucleic acid directly from the specimen

32 $$$$$$$$$$$$ Is it worth the cost? Rapid results Detection of a broader range of pathogens than what is practically possible with the use of traditional methods Cost savings from providing quicker, appropriate treatment?

33 Manufacturer must recoup high development costs by charging a lot for the new diagnostic product In time, IF THE TECHNOLOGY HAS SIGNIFICANT ADVANTAGES, competitive products emerge Competition leads to better-designed products with lower prices In time, the new technology, or a variant of it, becomes the standard of practice

34 Thank you