Lecture 8: QA/QC and Molecular Microbiology Part 1

Regulations and Governing Bodies:

• Federal law governs implementation, usage and quality programs the surround testing of human specimens

  • Clinical laboratory Improvement Amendments - Regulations passed down by CMS (1988 - known as CLIA ‘88)

  • Joint Commision (TJC), local state laws (state DOH), and other accreditting organizations (CAP, AABB, etc)

  • Some states have additional requirements for testing/accredidation (CLEP - Clinical Laboratory Evaluation Program, NYS DOH)

• Accrediting bodies will sometimes defer to whichever one is “stricter” regarding rules (CLEP > CAP > CLIA)

What do these “bodies” govern/say you have to do?:

• EVERYTHING

• Laboratory Safety/Information Systems

• Test Method Validation/Verification

• Standard Operating Procedures and Training

• Proficiency Testing

• Personnel Requirements/Competency of Staff

• Quality Control and Analysis

• So much, much more

Requirements for Testing/Reporting:

• Must be accredited first (apply for license to do the testing)

• Must be enrolled in a proficiency testing (PT) program (blinded challenges)

• Must undergo periodic review by other labs/teams (biannual insepction)

• Must have staff that are qualified and certified to run the tests alongside ALL THE PAPERWORK

• CAP - biyearly inspection where teams come in to review lab (files, reports, licensing, etc)

  • The guidance documents tell all…

Requirements for Testing/Reporting: Checklists:

• All Commons - All labs

• Specialist section (Micro, Chemistry)

Requirements for Testing/Reporting: What’s inside?:

• The thing you must do

• What thy expect you to do (why)

• How you have to keep track

Requirements for Testing/Reporting: This covers:

• Training and competency, safety, SOPs, IT stuff, testing, QA/QC and any new technologies

A new molecular test for TIKTOKVIRUS has been made - it’s been infecting people’s brains and zombifying them: How do we do it?:

• Decide what type of test to bring on (IVD v. others)

• How to operate the test (SOP)

• A way to show that the new assay is working correctly (blinded challenges — proficiency testing)

• Data showing that the test works great in your hands (validation/verification)

• A way to show that it continues to perform well and safely (QC plan)

IVD (In vitro diagnostic):

• “…products are those reagents, instruments, and systems intended for use in diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended fro use in the collection, preparation, and examination of specimens taken from the human body

• These assays are the easiest to show performance on and introduce

• Everything is made by the company. submitted to FDA and they tell you how to do it, what to test on, and how the test performs

  • Cant deviate from manufacturer’s instructions

Pre-market approval (new diagnostic):

• Goes through initial submission of safety and efficacy of new device in chosen field

510k Premarket notification:

• Comparative analysis of new assay to “gold standard” (one that did PMA), showing “substanial equivalency”

Emergency Use Authorization:

• Classification for emerging disease/infectious agents — limited FDA submission data (rare)

Laboratory Developed Test (LDTs):

• Developed by the lab (nothing on the market)

• Need to show that it works well (validation)

• Vendors won’t help (no tech support)

• Have to state “not FDA approved” and that “characteristics were determined by XXX laboratory”

• May be cheaper than IVD (can use equipment you already have)

SOPs (Standard Operating Procedure):

• Tell people how to do the test — new tests all need an SOP/manual

• Must include principle of test, what specimens it can be run on, rejection criteria, TAT, detailed protocol, reagent/instruments, results (expected), troubleshooting, QA/QC and limitations

• Must be signed by laboratory director and everyone who does the test

Proficiency Testing/Challenges:

• Regulated analytes require annual enrollement (CLIA defines)

• CMS tell us which ones are approved (PT 3× 5 samples/year)

• For non-regulated analytes (those not covered by CMS), lab has to test 2x per year

  • There are programs that will send you challenged 2x/yr

• If no PT is available, can work with other labs (peer/alt review) and show performance (blinded samples)

  • Alternative proficiency

Showing your test works: Validation/Verification:

• IVD requires a verification (unless modified)

• LDT requires a full validation

Verification:

• Shows that the assay works just like the manufacturer says

• FDA-cleared or approved tests only

• Checks accuracy, precision, reportable range, and reference range

• Compare new method to one already verified (test using same samples) — do this with ALL specimen types

• Any changes require FULL validation (don’t deviate)

Validation:

• Checks accuracy, precision, reportable range, and reference range

• ALSO checks for interferences, sensitivity, specificity, LoD, LoQ, etc

• Check against another method (IVD or LDT) that’s approved

• Define: Controls, protocol, and performacne chars

Qualitative tests:

• Require (1) Positive, (2) Negative analytes and (3) Non-template control (NTC)

• These show (1) assay detects what it should, (2) no false positive amplifications, and (3) no contamination

Quantitative tests:

• Require (1) High and Low positive, (2) Negative, (3) Non-template control

• Have a “standard curve” of known analytes and specific concentrations

External Control:

• Typically bought by someone other than the test maker

• Previously tested (pos/neg) samples work too

• Shows assay detects targets/works

Internal control:

• Typically part of the assay (checks for quality)

• Can be human genes or spiked in target

  • beta-globulin gene in HPV endocervical samples

  • Bacillus spores in test for C. difficile

• If does not amplify, cannot report — INVALID test

Extraction control:

• Used to show that nucleic acid extraction worked

• Can be same control as “internal” or “external”

• Many companies will use this as a schema to save on reagent costs (fewer tubes to run and less ctrl material to use at once)

Why do we need so many controls?:

• Manufactuers define conditions in IVD approval as to how many controls need to be run

  • Typically defined within a 24 hr window or per each “batch” of tests

• If LDT, control frequency must be defined by the lab

• Some tests do not have defined frequency to be run, thus the labe must work on defining this (ICQP)

• All control performance must be tracked (logged and tracked for compliance)

  • This must be done prior to reporting ANY specimen results

IQCP: Individualized Quality Control Plan:

• QC plan that allows for decreased control material utilization (weekly or monthly) IF the assay allows for it (instructions)

  • In NYS, no less than 1 pos sample/mon, 1 neg sample / week

• Each labe must carry out risk assessment and review of performance (20-30 d of runs)

  • Monitored monthly following approval — signed by Lab Director

  • Changes/updates needed if deviates from prior approval conditions (temp change, increase in false positives, instruments non functional, etc)

  • Used to cut down on usage of individual cassettes / reagents (cost)

Molecular Microbiology:

• Detection, identification and analysis of microorganisms

  • DNA or RNA

Advantages of Molecular Microbiology over traditional microbiology (culture, stains, and biochemical testing):

• Rapid turnaround time

• Better sensitivity and specificity

• Can be quantitative

• Comparison of biochemically similar organisms (epidemiology)

Why care about Molecular Microbiology?:

• Molecular testing is increasingly common

• Unlike genetic screening or oncology, microbiology testing is relatively widespread

  • EVERYONE gets sick, multiple times throughout their life

• Rapid advance in technology enables us to detect previously unknown pathogens (or rarely ID’d organisms)

  • Legionnaire’s Disease (L. pneumophilia), COVID-19 (SARS-CoV-2)

Molecular testing can drastically impact patient care:

• Improved patient care (decreased mortality, morbidity, and length of stay)

• Lower costs to the hospital system

• Avoid unnecessary antibiotic administration (or decrease utilization of broad spectrum antibiotics)

• Lower infection rates by identifying people colonized by multidrug resistant orgnisms

Applications of Molecular Assays in Microbiology:

• Molecular assays have in almost all labs replaced viral culture:

– Rapid / high-throughput differentiation of viruses (DNA, RNA) that

cause similar syndromic conditions

• Antibiotic resistance testing for tailored antimicrobial therapy

– Change drug choices when they have a resistance marker (e.g. tetM

might not use tetracyclines)

• Quantification of viruses (or other organisms) in specimens

– HIV viral loads

• Genotyping, classification, and epidemiological studies

• Discovery of novel pathogens

– Sequencing discovery of SARS-CoV-2

• Microbiome / Virome / Mycobiome studies

– How changes in our normal flora relates to disease (more research)

Signal Amplification Methods:

• Normally use a nucleic acid probe combined with some form of amplifying signal producer (enzyme):

  • Branched-chain DNA

  • Hybrid Capture

  • In situ hybridization

• Typically considered less sensitive than NAATs

• Less likely to be contaminated than NAATs (false positive results)

Fluorescent ISH:

• Uses hybridized probes of DNA (or peptide, PNA) to identify intact microorganisms:

  • Bind to rRNA molecules on microbes (higher copy number than a single gene) and aid in ID/selection of antimicrobials

Accelerate Pheno System:

Nucleic Acid Amplification Tests (NAATs): qPCR/RT-qPCR dominates most NAATs

• Predominant workhorse of most molecular diagnostic labs

• End stage PCR (gel electrophoresis, PFGE, etc) has been mostly replaced by qPCR

SYBR Green:

• Primers specific for a region of interest (ROI)

Taqman:

• Primers and an ROI specific probe

Principles of NAATs (e.g. qPCR) for pathogen detection:

• DNA (or RNA) extraction → Target sequence amplification with qPCR (or RT-qPCR) → Simultaneous detection of products w/ fluorescent probes

NAATs: Qualitative assays Presence or Absence of a gene/ROI:

What are Qualitative Assays good for?:

• Diagnosis of infections:

  • SARS-CoV-2

  • Influenza A/B

  • Human Papillomavirus (HPV)

  • HSV1, HSV2, VZV

  • Mpox Virus (MPXV or Ortopoxviridae)

  • Many others

Syndromic-based diagnostic panels:

• Test for multiple pathogens in one test:

  • Respiratory panel

  • Gastrointestinal Disease Panel

  • Meningitis/Encephalitis Panel

  • Blood Culture ID Panel

• Many of these are “sample-to-answer” platforms where assay complexity is highly reduced:

  • Nucleic acids extracted onboard

  • PCR carried out on cartridge

  • Self-sealed containers than can be discarded once used

NAATs: Quantitative assays:

• Uses standard curve (serial dilution of known concentrations) run in parallel to allow for estimation of titer within a sample

• Used for diagnosis/tracking of titers of organisms in bodily fluids:

  • namely viral titers in serum/plasma

  • Ex:

  • Human Immunodeficiency Virus-1

  • Hepatitis C Virus

  • Hepatitis B Virus

Quantifiable range vs reportable range:

• Not always the same

• Things can be positive < or > your limit of quantitation (LoQ), and are still positive

• You cannot report outside of the LoQ (only that it’s positive)

• Ct values =/= quantitation (and should not be interp that way)

NAATs: Modifications of PCR:

• Different assays use modified protocols or methods within the “PCR” realm for augmenting specificity or allowing for greater number of analytes per test:

  • Broad range PCR

  • Nested PCR (Biofire plateforms use this technology)

  • Multiplex PCR (commercial platforms with 2+ targets use this)

  • Reverse Transcription PCR (RT-PCR) is used by assays with targets that originate as RNA (HIV, HCV, etc)

How do you select target sequence for identification of microorganisms?:

• Genomic or plasmid DNA, genomic RNA

  • Unique gene/sequence for the pathogen of interest (e.g. virulence factors)

  • Antibiotic resistance gene

  • For viruses with various subtypes, use:

    • Sequence shared by all types for initial detection

    • Use type specific sequence for further typing

Major considerations of using molecular tests:

• Cannot differentiate between alive/dead organisms

  • Remnant nucleic acid can persist for weeks or even months

  • Many PCRs should not be used as “test of cure” for this reason

• Not all tests and targets are equal - know test limitations

• Not detected =/= not present

  • Pathogen under detection limit, wrong location, etc

• Genotype =/= Phenotype

  • Presence of antibiotic resistance gene does not mean the bacteria is resistant

Clostridium difficile:

• Gram positive, anaerobic, spore-forming bacillus

• Spores: critical for prolonged survival in the environment and ability to spread

• Causes 15-25% of all antibiotic-associated pseudomembranous colitis (diarrhea)

• In recent years healthcare associated diarrhea has increased in incidence and severity

• C-difficile infections are associated with an increased hospital stay, morbidity, and mortality among patients

C. difficile Pathogenesis:

• Antibiotic therapy → C. difficile exposure and colonization → Toxin production → Effective immune response = asymptomatic, Inadequate immune response = diarrhea

Pathogenicity locus of C.difficile: PaLoc

Glutamate dehydrogenase (GDH) EIA:

• Fast and sensitive, but not specific (false-positives)

• All C.difficile strains (toxic or not), other clostridial species and other bacteria produce GDH

Clinical Impact of an Ineffective Test:

• Repeating the tests

• Delay with prescribing an antibiotic against C.diff

• Increase in severity/deterioration of the illness

• Increase the length of stay

• Time before isolation > exposure of other patients > transmission

• Increase in number of infections

• NEED A TEST FOR C.DIFFICILE DETECTION WITH MORE SENSITIVITY AND QUICK TURN AROUND TIME

C.Difficile detection by multiplex qPCR w/internal control:

Internal Control for C.diff assay:

• Add an internal control in each sample

  • spores of Bacillus globigii

  • Note: internal control tests the validity of each sample

• Postive I.C

  • The nucleic acid extraction worked

  • Also checks presence of PCR inhibitors

  • Valid test

Negative I.C. C.difficile:

• Invalid test

• Sample needs re-extraction

qPCR detection of C.difficile using GeneXpert (Cepheid):

• Quick TAT: 50 min

• Easy set up, automated (no highly trained personnel required)

• Closed system (less contamination)

• Detects toxB gene and internal control

• High sensitivity (>94%) and specificity (>94%), NPV (98.8)

• Expensive!

GeneXpert Assay: Inside the cartidge:

• Closed system

  • Less contamination

  • Could be done in other sections of the lab

• Result:

  • C.diff positive or negative

Current Epidemic Strain of C.difficile:

• Increasing incidence and severity

• Outbreaks of severe disease by the epidemic strain:

  • BI/NAP1/027, toxinotype III

  • Resistant to fluoroquinolones

  • Increased spore production

  • Carries extra toxin known as binary toxin (cdtB, cdtA)

  • Increased toxin A/B production due to a polymorphism in the regulatory gene (tcdC)

Pathogenicity Locus of C.difficile NAP-1/027:

• Binary toxin is coded for by cdtA and cdtB genes outside PaLoc

• 1 bp deletion in tcdC inactivates repressor of tcdB

  • Increased tcdB transcription, i.e. Toxin B production

Positive Toxin B: Presumptive Positive B1/NAP1/027