QA/QC and Molecular Microbiology Part 1

Regulations and governing bodies

• federal laws governs implementation, usage and quality programs that surround testing of human specimens

  • Clinical Lab Improvement Amendments - regulations passed down by CMS (CLIA ‘88)

• Joint commission (TJC), local state laws (state DOH), and other accrediting orgs (CAP, AABB)

  • some states have additional requirements for testing/accreditation (CLEP - clinical lab eval program, NYS DOH)

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

So what do they tell us how to do the tests?

• must be accredited first (apply for license)

• must be enrolled in PT program (blinded challenges)

• must undergo periodic review by other labs (biannual inspection)

• must have staff that are qualified and certified to run tests alongside ALL PAPERWORK

• CAP - biyearly inspection where teams come in to review lab

Requirements for testing/reporting

• whats inside?

  • thing you must do

  • what they expect you to do (why)

  • how you have to keep track

• This covers

  • training/competency, safety, SOPs, IT, testing, QC/QA

IVD (in vitro diagnostic)

• intended for use in diagnosis of disease or other conditions

• easiest to show performance and introduce

• use in collection, prep, and examination of specimens taken from human body

• everything made by company therefore cannot deviate from manufacturer’s instructions

Pre-market approval (new diagnostic)

goes through initial submissions of safety and efficacy of new device in chosen field

510k Premarket notifcation

comparative analysis of new assay to “gold standard” showing substantial equivalency

Emergency use authorization

classification for emerging diseases/infectious agents - limited FDA submission data (rare) [ex. COVID testing]

Laboratory developed test (LDTs)

• developed by lab (nothing on 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 X lab”

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

Proficency testing/challenges

• regulated analytes require annual enrollment (CLIA defines)

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

• for non-regulated analytes, lab tests 2x/yr

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

Showing your test works

• IVD requires verification (unless modified)

• LDT requires full validation

verfication

• shows assay works like 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 w ALL specimen types

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

Validation

• checks accuracy, precision, reportable range, reference range

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

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

• define: controls, protocol, and performance chars

Qualitative tests

• require +, - analytes and non-template control (NTC)

• these show assay detects what it should, no false pos amplifications, and no contamination

Quantitative test

• require (1) high and low pos, (2) -, (3) NTC

• have “standard curve” of known analytes at specific concentrations

External control

• typically brought by someone other than test maker

• previously tested (post/neg) samples work too

• shows assays detects targets/works

Internal control

• typically part of the assay (checks for quality)

• can be human genes or spiked in target

• no amplification = can’t report = INVALID test

Extraction control

• used to show that nucleic acid extraction worked

• can be same control as internal/external

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

Why do we need so many controls?

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

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

• if LDT, control frequency must be defined by lab

• some tests don’t have defined frequency for QC to be run, thus lab must work on defining this (ICQP)

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

  • must be done prior to reporting ANY specimen results

IQCP: individualized quality control plan

• QC plan that allows for decreased control mat utilization IF Assay allows for it

  • in NYS no less than 1 + sample/mon, 1 neg sample/week

• each lab must carry risk assessment and review of performance (20-30d of runs)

  • monitored monthly following approval - signed by lab director

  • changes/updates needed if deviates from prior approval conditions

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

Molecular microbiology

• detection, ID, and analysis of microorganism (DNA/RNA)

• advantages:

  • rapid turn around time

  • better sensitivity and specificity

  • can be quantitative

  • comparison of biochemically similar organisms (epidemiological)

applications of molecular assays in microbiology

• replaced viral culture in most labs

• antibiotic resistance testing tailored for antimicrobial therapy

• quantification of viruses

• genotyping, classification, and epidemiological studies

• discovery of novel pathogens

• microbiome/virome/mycobiome studies

DNA detection methods

• signal amplification methods

• nucleic acid amplification tests (NAATs)

• post-amplification analysis methods

• sequencing and NGS

Signal amplification methods

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

  • branched DNA

  • hybrid capture

  • in situ hybridization

• typically less sensitive than NAATs

• less likely to be contaminated than NAATs (false +)

In situ hybridization (Fluorescent -ISH)

• use hybridized probes of DNA (or peptide, PNA) to ID intact microorganisms

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

Nucleic acid amplification tests (NAATs)

• qPCR or RT-qPCR

• predominant workhorse of most molecular diagnostic labs. End-stage PCR have mostly been replaced by qPCR

• SYBR green (primers specific for ROI) and TaqMan (primers and an ROI specific probe)

region of interest - ROI

qPCR test principles

NAATs: qualitative assays

• presence or absence of a gene/ROI

what are qualitative assays good for?

• diagnosis of infection

• syndromic-based diagnostic panels (test for multiple pathogens in 1 test)

examples of syndromic-based panels

• many are “sample-to-answer” platforms where assay complexity is highly reduced

  • nucleic acids extracted onboard

  • PCR carried out on cartridge

  • self-sealed containers that 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

• quantifiable range and reportable range are not always same

• things can be + < or > limit of quantitation, and are still +

  • cannot report outside of LoQ (only that it is +)

  • Ct values =/= quantitation (and shouldn’t be interp that way)

How do you select target sequence for ID of microorganisms?

• genomic or plasmid DNA, genomic RNA

  • unique gene/sequence for pathogen of interest (virulence factors)

• antibiotic resistance gene

• for viruses with various subtypes, use:

  • sequence shared by all types for initial detection

  • use specific sequence for further typing

Major considerations of using molecular tests

• CANT differentiate between alive/dead organisms

  • remnant nucleic acid can persist for weeks or even months

  • many PCRs shouldn’t be used as “test of cure'“

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

• not detected=/= not present

  • pathogen under detection limit, wrong location

• genotype =/= phenotype

  • presence of antibiotic resistance gene doesn’t be bacteria is resistance

C. diff

• pathogenicity locus: PaLoc

  • produced by tcdB and tcdA [produce toxins B and A]

    • most assays try to target tcdB

Internal control for C. diff assay

• IC: spores of Bacillus globigii

  • tests for validity of each sample

• + IC

  • nucleic acid extraction worked

  • also checks absence of PCR inhibitors

    • valid test

• - IC

  • invalid test

  • sample needs re-extraction

qPCR detection of C.diff using GeneXpert (Cepheid)

• quick TAT: 50 min

• closed system

• detects ToxB gene and internal control

• high sensitivity and specificity

severe epidemic strain of C. Diff

• BI/NAP1/027, toxinotype III

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

• increased toxin A/B production due to polymorphism in regulatory gene (tcdC)

Glutamate dehydrogenase (GDH) EIA

• fast and sensitive, but not specific (false +s)

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