Dept. Diagnostic MedicineSMBD-JewishGeneralHospital, Montreal, Qc

 

New method for determining real C.difficile infections by detecting the total bacterial load in stool samples using

the universal bacterial 16S rDNA qPCR assay

Ivan Brukner Ph.D. Lady Davis Institute for Medical Research

Key words: total bacterial load, stool samples, qPCR assay, normalisation

OutlineLimitations of qualitative PCR as a tool for precise

diagnosis of infections caused by opportunistic pathogens

Method proposed by JGH in collaboration with McGill University

ResultsConclusionsAdvantages of the Method for Focus DiagnosticsCurrent IP StatusSuggestions for next steps

Limitations of qualitative PCR as a tool for precise diagnosis of infections caused by opportunistic pathogens

Opportunistic pathogens:  Infectious agent that can only cause disease when the host's resista

nce is low Found in stool, urine, respiratory samples

Ex: C.difficile, H.pylori, Haemophilus influenzae, Streptococcus pneumoniae

Qualiative PCR : Detects the presence or absence of a certain sequence Does not distinguish samples of symptomatic vs non symptomatic

patients with opportunistic bacteria in their microbial flora Ex.: Detection of C.difficile: Current PCR methods targeting Toxin B or Toxin

A and B can not distinguish with certainty the sample of a patient who has CDI from someone who is a C.difficile carrier but has a liquid stool due to either drugs, another pathogen or laxatives.

How can we improve the current method for diagnosis of infections caused by opportunistic pathogens?

We need to determine the ‘threshold’ of concentration for different pathogen of interest in total microbial flora which will allow us to determine when an opportunistic pathogen otherwise not causing any symptoms becomes pathogenic and can thus cause an infection. Determined for each pathogen by collection non symptomatic patients

Patient with symptomsRelative concentration>= Threshold

Carrier ( asymptomatic patient)Relative concentration< Threshold

Threshold of relative concentration =

lowest amount of opportunistic pathogen to cause symptoms total amount of microbial flora

Why does mass normalisation not work with liquid stool?

Because swab with SAME MASS can be taken from

person having 10 diarrhea per day (each ~200-400ml volume in total) or from carrier (normal stool)

Stool dilution effect is making normalisation based on mass impossible to estimate clinical impact of Ct tox B or A with Ct>=30

Method proposed by Jewish General Hospital (JGH) in collaboration with McGill University

A novel method that allows one to determine the threshold of relative concentration for various opportunistic pathogens in total microbial flora. Values bellow the threshold would be relevant for asymptomatic carriers.

Method which will allow for the adequate normalisation of analytical signal This can be done only if one takes into account total microbiota

(ex: 16S universal)

Next (inevitable) step : Quantitative microbiology?

Our Methodology for detecting total bacterial load in C.difficile samplesSample study size: 500 liquid C.difficile positive

stools and 50 formed C.difficile negative stoolsStudy preliminary period: January-October 2014Identification of patients for C.difficile screening

was performed on admission according to established hospital infection control policies.

For measurements of wet stool mass entering the PCR reaction, 49 stool samples were randomly chosen and processed.

Stool example and C.difficile

There is no correlation between clinical sample mass (stool) and total bacterial load

Total bacterial load is determined by more dominant parameters (not mass)Ex: drug uptake, infection status/diet/immune status…

What type of signal normalisation is important for detection of opportunistic pathogens?

Relative normalisation using total bacterial load in microbial flora The same Ct value (ex: Ct>=32) for either Toxin A and/or toxin B

could have completely different clinical meaning if the context of total bacterial flora is different (16S qPCR Ct=12 versus Ct=22)

Summary of Results

The quantity of stool material attached to swab during “typical” swabbing process had an average value and standard deviation of 16.5 (+/- 9.6) mg

“Average” stool has 1012 bacteria/gram The average number of bacteria entering direct qPCR assay is

~107-108. In individual samples, the clinical variability of total bacterial load

per swab deviates from average numbers by spanning 3 log (10) values range. These deviations could have significant clinical meaning, especially in the

cases where antibiotic and/or infection-induced changes in micro biota could be relevant for diagnosis and therapy

Comparison of bacterial load measurement techniques

Figure 1. Stool sample mass (mg of stool entering sample buffer) and Ct value of 16S rDNA universal PCR assay (R2 = -0.12, P= 0.58)

Stool mass

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Total bacterial load varies between individuals and it does not correlate with the stool mass

Comparison of bacterial load measurement techniques Figure 2. Binned total bacterial counts versus Ct values of 16S rDNA qPCR assay done with stool swab lysis protocol (Ct values above 18 have low bacterial load and should be considered as samples with strongly reduced micro flora).

There IS correlation between total number of bacteria in stool sample and Ct values of our assay

logN

Ct

Variability of the total bacterial load among clinical samples

Figure3. Distribution of Ct values amongst 500 stool swabs using our 16S qPCR assay

Ct values of the 16S assay

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(to

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How much total bacterial load vary among clinical samples (C difficile positive)?1000x !!!!!!

LOR LOD Average efficiency (SD) r2

Nadkarni and modifications* 1000-10E8 1000 97% (2%) >0.994

mod Clifford 10000-10E8 1000 94% (4%) >0.961

Typical RFU versus cycle diagram for stool sample

Figure 4. The 10x serial dilutions of DNA isolated from stool. Excellent correlation for both assays is in range of 12-24 Ct units

Zone masked by ‘contamination’

Reference PCR (Nadkami and modifications)

JGH 16s qPCR( based on Modified Clifford method)

LOR: Linear Operative Range of the assay ALOD: Average Limit of Detectionr2-value: Correlation Coefficient SD: Standard Deviation

Conclusion

New short qPCR for 16S rDNA has the ability to measure total bacterial load in the clinically relevant range

Advantages of the new qPCR (a) the only existing PCR assay with a short amplicon for 16S rDNA

(~200bp) compatible with hydrolysis probe technology (TaqMan-like) and direct PCR (no nucleic acid isolation)

(b) does not show any self-priming/ false priming, thus allowing confident resulting

Next step: Collection of ~100 non-symptomatic C diff positive samples in order to establish the threshold which will allow one to determine when opportunistic pathogen starts to cause symptoms.

Advantages for IVD INDUSTRY in obtaining and incorporating this method into its current

assays

Compatibility with present qPCR chemistry (TaqMan-like)

Early IP ownership and advantage over competition (not simple to design another 16S universal assay with such good performance, with

short amplicon and without self-priming/cross-reaction)

Covering/controlling IVD diagnostics of opportune pathogens in all microbiota samples with this IP (stool: C difficile, VRE…respiratory:

Streptococcus Pneumonia…,urine: VRE…

Current IP status

• US provisional filed January 13th, 2014 and will be converted into a PCT in January 2015

• Current title of the IP: METHODS, REAGENTS AND KITS FOR THE ASSESSMENT OF CLOSTRODIUM DIFFICILE INFECTION

• Authors: Andre Dascal, Yves Longtin, Matthew Oughton, Ivan Brukner

Possible steps going forwardCollaboration with the team to advance this assayLicensing the technology through the purchase of the patent from JGH