self-swabbing sample adequacy control PCR qPCR molecular diagnostics pathogen disease health fast testing

1. Rectal swab screening assays of public health importance in molecular
diagnostics: Sample Adequacy Control
Sanja Glisovic, Shaun Eintracht, Yves Longtin, Matthew Oughton and Ivan Brukner
http://www.jiph.org/article/S1876-0341(17)30183-1/fulltext

2. Sample Adequacy Control (SAC)
What is it?
The Sample Adequacy control is a “built-in check” to ensure that the input sample is
adequate for testing. Usually, an endogenous human gene is co-amplified to ensure
adequate sample has been collected.
What does it do? The SAC verifies that there is sufficient material to ensure confidence
in a negative result.
A negative SAC indicates an INVALID result
Note: our molecular assays have SAC (nasal, skin/mucal swabs)

3. For nasal and/or genital swabs->
single copy human gene is “normal”
part of the assay (RNase P example)
For rectal swabs - there is (frequently) not
enough human DNA
(although if all swab material will be used ->
it might
be enough material for human SAC, but
number of “empty”
swabs might be too high for clinical
applicability)
Currently: Rectal swab are passing “visual”
Quality Control

4. Is1
Is the swab visibly soiled?
2
In order to escape a false negative, better
cancel the swab,
- It will be re-done in 48 hours minimum,
or 2-3 weeks maximum, by our ULTRA
FAST hospital screening method
LABORATORY
Rectal swab

5. Days required to re-swab 241 cancelled samples during one month (May 2016): the
x-axis presents the number of days required for the next admissible swab to be
received, while the y-axis is the number of admissible swabs resubmitted following
the delay specified along the x-axis.
http://www.jiph.org/article/S1876-0341(17)30183-1/fulltext

6. Among 241 cancelled swabs, 16 had to be recollected more than three times due to the
repeated absence of colored fecal material, and two patients required rescreening seven times
each until an admissible sample was obtained.
Is this criteria even reliable?
The underestimation of sample adequacy has been recorded in work done by Curry et al. (J Clin
Microbiol. 2011 Nov; 49(11): 3788–3793, see “Swab visibly feculent” on Table 5). They showed
that 15 out of 26 swabs which are not visible soiled, where nonetheless positive for toxB of C.

7. 15$2
Commercial ULTRA FAST molecular screening is ~
15-25$ per patient in emergency, “only” 10%
have to be re-tested in 48h-2 weeks, but 90% will
have fast “answer”
Is our new ULTRA
FAST hospital -
screening
method cost-
effective?
Yes, we fired our PhD and 2
molecular technologists and
adopted idiot-proven
automatic screening method

8. Rectal swabs (BBL CultureSwab, BD) were collected from 717 healthy volunteers after confirming their health
status between November 2016 and February 2017. Inclusion criteria included: no systemic antibiotics for a
minimum of 2 weeks prior to enrolment; absence of gastrointestinal symptoms; and the absence of probiotic
supplements usage at the time of enrolment. To minimize subjective bias of “visual estimation”, absence of
visible fecal material was not used as a criterion for swab rejection. The protocol for this study was reviewed
by our institution’s Research Ethics Committee.
http://www.jiph.org/article/S1876-0341(17)30183-1/fulltext

10. Distribution of Cq values generated by RNase P and 16s rRNA qPCR assays using DNA isolated from
rectal swabs of healthy population. The 3 data values moving average was used to produce 16s rRNA
and RNase P curves (solid lanes) from “frequency versus Cq” experimental values (illustrated by circles
and triangles, respectively); 16s rRNA (Cq mean/SD) = 21.13/3.1; N = 713; RNase P (Cq
mean/SD) = 32.74/2.8; N = 527.
http://www.jiph.org/article/S1876-0341(17)30183-1/fulltext

11. Our SAC (16S), was estimated by using only 1/100 fraction of sample
material. Hundreds of different screening qPCR reactions can be
performed from the same sample and only 0.4% of 16S assays was
show to be “invalid”. By setting an arbitrary cut-off value of Cq=27.7
for the 16s rRNA assay, only 2% of swabs (DNA isolation protocol)
would be considered “invalid”, comparing to ∼10% canceled in clinical
screening, by “visible soiled criteria”.
http://www.jiph.org/article/S1876-0341(17)30183-1/fulltext

12. Assay for estimating total bacterial load: relative qPCR normalization of bacterial load with associated clinical implications:
hidden beautiful science behind SAC: quantitative molecular microbiology
DOI: http://dx.doi.org/10.1016/j.diagmicrobio.2015.04.005
A: Absence of correlation among wet mass
(mg) of stool samples and the value of Cq
of 16S rDNA qPCR, using different stools
(“biological variability effect”)
B: Presence of correlation among different
methods of DNA isolation among the same
samples
C: Presence of correlation among
total anaerobic bacterial numbers of
different stool samples, as measured by
colony counts and 16S rDNA qPCR, using
different stool samples

13. What is the price of centralised diagnostic harmonisation if the
quality of service is in question?
Once implemented, can it be undone/changed? How easily?
Is there a value in diverse approaches?

15. FUTURE: Evaluation of self-swabbing coupled with a telephone
health helpline as an adjunct tool for surveillance of influenza
viruses in Ontario
How long is a centralised screening method going to be acceptable from the point of
assay quality, if sample adequacy control is not introduced?
For example: samples from remote location? Sample integrity and quantity?
What damage is going to be done by delaying screening assay for 48h-2 weeks?
What will happen when better quality assays emerge?

16. Why is all of this important:
(A) control of infections
In epidemiology, the basic reproduction number (sometimes
called basic reproductive ratio) of an infection can be thought of
as the number of cases one case generates (on average) over the
course of its infectious period, in an otherwise uninfected
population.

17. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653956/pdf/ofv15
2.pdf
http://jmm.microbiologyresearch.org/content/journal/jmm/10.1099/
jmm.0.000381
Self-swabbing and postage of nasal swabs prior to processing has no
effect on yield of S. aureus, and could facilitate large community-
based carriage studies.
(B) Simplicity with lower cost
Self-swabbing …even from home…

18. ( c) Win-win for all
”
Possibility to decrease hospital visits (swabbing from home) –
Patient is happy
Allowing bigger “screening volume” and generating more positives
MD is happy
Protocol is overall cheaper (TAT faster, removing duplicated swabbing)
Ministry of Health should be happy?
And as collateral “damage”: Post Canada has more job openings (happy too)
For more details: good source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101344

19. C difficile 1.07-2.6
Assessing the effect of patient screening and isolation on curtailing Clostridium difficile infection in hospital settings
Sara MaghdooriEmail author and Seyed M. Moghadas BMC Infectious DiseasesBMC series – open, inclusive and trusted (2017) 17:384
https://doi.org/10.1186/s12879-017-2494-6

20. • Which infective organisms can be typically screened by rectal swab…
• Bacteria
• Vibrio cholerae (cholera); Clostridium difficile (pseudomembranous enterocolitis);
Shigella (shigellosis / bacillary dysentery); Salmonella typhii (typhoid fever); Vibrio
parahaemolyticus; Escherichia coli; Campylobacter
• Viruses
• Hepatitis A; Hepatitis E; Enteroviruses; Norovirus acute gastroenteritis; Poliovirus
(poliomyelitis); Rotavirus
• Protozoans
• Entameba histolytica (amoebiasis); Giardia (giardiasis); Cryptosporidium
(cryptosporidiosis); Toxoplasma gondii (toxoplasmosis); Helminths; Tape worms;
Ascariasis and other soil transmitted helminthiasis

21. “Do it yourself”

23. If SAC is correct, even cotton
swabs work

24. Matthew Yves
SanjaShaun
Nadia
Thank you for “usual” help: Stacy and Coleen…concept of SAC: Andre Dascal…