NEW DEVELOPMENTS IN RAPID

DIAGNOSTIC TECHNOLOGY FOR

ANTIMICROBIAL RESISTANCE

E.C. Alocilja1, PhD, Professor

Co-authors:

M. Vasher1, B. Etchebarne1, Z. Li1, C. Gordillo2, A. Gomez2, H. Sanchez2, R.

Bhattarai3, N. Bhusal4, R.K. Briceno5, S. Benites5, L. Jyothi6

1Michigan State University, USA; 2El Colegio de la Frontera Sur, Mexico; 3Agriculture and Forestry University, Nepal; 4Kathmandu University, Nepal;

5Universidad Cesar Vallejo, Peru; 6MediCiti Institute of Medical Sciences, India

12th OIE Seminar and 18th World Association of Veterinary Laboratory Diagnosticians Symposium

Sorrento, Italy, 7-10 June 2017

Presentation Outline:

1. Overview of antimicrobial resistance (AMR)

2. Nano-biosensor triage and detection technologies for AMR monitoring

3. Data integration and global biosurveillance of AMR

Presentation Outline & Result Highlights

Highlight of Results:1. Triage nano-biosensor (TNB) provides quick phenotypic screening:

- Simple to use: 3-step process- Affordable and accessible: < $0.05/test- Rapid: < 10 min- Antimicrobial susceptibility testing- Actionable outcomes: guides antibiotic use, allocates resources

2. PCR-less DNA biosensor provides specific test- Specific- Affordable: < $1/test- Rapid: ~2 h (from lysis to detection)

3. Data integration- Phenotypic, genomic, syndromic, historical data- Global biosurveillance of antimicrobial distribution

Quick review

Pan-AmerianHub

Asian Hub

Australian Hub

European Hub

African Hub

Overview of AMR Integrated Nano-Biosensor Technology

FIRST: Field-operable, Inexpensive,

Real-time data analysis, Sensitive &

specific, Trend generation

Biosensor schematic

Global

LocalHumans

Animals

Wildlife

• Triage & Specific Diagnostic Technologies

• Global Biosurveillance System

• Appropriate for Low Resource Settings

Flowchart for the Triage, Diagnostic & AMR Monitoring

Triage Technology

Active case finding of AMR in humans, animals, and food chain

NegativesPositives

Confirm disease absence

Confirm disease presence

Intervention: treatment, therapy, etc.

Step 2: Diagnostic DNA biosensor - to determine specific disease

Step 1: Triage matting - to determine sick/not sick

Step 3: Monitor treatment efficacy by matting – determines AMR case

A. Biosensor Technology

Triage

Diagnose

Monitor

Data from triage & biosensor

Schematic of the biosurveillance map at the community level

Schematic of the biosurveillance map at the global level

Environmental data & other data sources

Issue alerts

Prioritize resources

Implement control programs

Actionable outcomes community level

Flowchart for the Data Integration and Biosurveillance

B. Data Integration and Biosurveilance

Syndromic data

Issue alerts

Prioritize resources

Implement control programs

Actionable outcomes global level

Achieve goals:

• Prevent illness

• Save lives

• Improve quality

of life

• Reduce

healthcare cost

• Increase

productivity

• Reduce poverty

• Increase exports

• Increase

livestock &

animal

production

• Reduce AMR

Data from global partners

Let’s Begin: My Story on AMR

Lucia Vazquez, a Tzotzil from San Cristobal de las Casas, Chiapas, Mexico. Her father was sick with MDR-TB undergoing 1.5 years of treatment: 1 h away weekly, 7 h away every 2 months.

A sign saying “I can stop TB” in various Mayan languages. There are only about 6 million Mayans in the world.

• Tuberculosis (TB): 2 billion people infected

• 8 million new cases per year

• #1 infectious killer worldwide

• 1.4 million deaths per year (~3/min)

• Co-morbidities: MDR-TB, HIV, Diabetes, Cancer

TB

Wildlife

Humans

Animals

Challenges in existing TB diagnostic techniques:

A TB test that is 85% sensitive and 97% specific can result in saving 400,000 lives annually (Desikan, 2013).

TB is a complex disease

Detect disease early while patient’s (animal, human) immune system is still high.

Economic and Global Implications of AMR by 2050

• > 5% loss of GDP for low-income countries

• 28 million more people into poverty

• 1.1% -3.8% shrinkage in volume of global real exports

• $300 billion - > $1 trillion per year global healthcare costs

• 2.6% - 7.5% per year decline in livestock production

“Unless addressed swiftly and seriously and on a sustained basis—the

growing global problem of antibiotic resistance will be disastrous for human

and animal health, food production and global economies”1.

Antimicrobial Resistance (AMR)

Examples of pathogens showing resistance to antibiotics:• Klebsiella pneumoniae – carbapenem• E. coli – fluoroquinolone• Gonorrhea – cephalosporin• Carbapenem-resistant Enterobacteriaceae – Colistin

1 World Bank Report

Drivers in Developing Rapid Diagnostics

Coinfections, Globalization

Vulnerable populations:

Immune-

compromised,

elderly, very young

Medical infrastructure

& diagnostic

capabilities

Drug resistance

Surveillance system

Wildlife

Humans

Animals

Promises of nanotechnology

Complexity of infections

Technologies:Simple Affordable Rapid

Pictures from the web

Env.

Overuse of antibiotics

Humans & Animals

Poultry industry in Bharatpur, Nepal

E. coli isolates:

- Colistin resistance (Mcr1) - 77.63%

- Nalidixic acid resistance - 60.53%

- Enrofloxacin resistance - 57.89%

Salmonella isolates:

- Multiple drugs (2 or more) - 73.68%

- Colistin resistant Salmonella in chicken meat

Antibiotic Use:

- 30% of antibiotic prescription is colistin sulphate

- 40% of colistin sulphate is used in feed supplement

Emerging AMR in the Food Chain

Drug resistance transmission in the food chain – from Bharatpur to Kathmandu

Case in Bharatpur, Nepal

Bacteria Number of isolatesMDR strains Total

MDRPercent

0 drug 1 drug 2 drugs >2 drugs

E. coli 40 0 7 5 28 33 82.50%

Multiple Drug Resistance (MDR) pattern of E. coli isolated from chicken meat

Bacteria Number of isolatesMDR strains Total

MDRPercent

0 drug 1 drug 2 drugs >2 drugs

E. coli 20 0 0 11 9 20 100%

Multiple Drug Resistance (MDR) patterns of E. coli isolated from human

tetA Sul1 QnrA

Chicken meat 62.50% 42.10% 0%

Human 70% 37.50% 0%

Distribution of antibiotic resistance genes in E. coli strains isolated from chicken meat and human

AMR in the Food Chain

1. Expensive

2. Fragile in harsh environmental conditions

3. Poor performance in complex matrices

4. Requires skilled personnel to operate

5. Requires a lab infrastructure

6. Requires electricity, refrigeration, sanitary area

7. Time-consuming

8. Phenotypic or genomic but not both

9. Not applicable in resource-poor settings

Constraints in Existing AMR Diagnostics

We need a paradigm shift in diagnostic development!

Acknowledgment: Pictures were taken from the web.

The diversity of genetic

mechanisms may exceed the

capabilities of current

molecular technologies to

detect resistance.

Current AMR susceptibility

assays take too long.

Introducing: Triage Diagnostic Technology

Concept from triage system in ER

Concept from population screening for disease risk

http://keywordsuggest.org/gallery/1222556.html

Triage nano-biosensor technologies and Triage methodology

Triage diagnostic technology (TDT): Higher sensitivity, even at lower specificity, is desired if disease is

lethal and early detection markedly improves prognosis

https://www.gov.uk/guidance/nhs-population-screening-explained

Paradigm Shift in Biosensor Development: Triage Approach

Triage Approach –impacts outcome when resources are low and demand for services is high.

Triage technology:High sensitivityRapidAffordableSimple

Diagnostic technology:High specificityHigh sensitivity

Triage Diagnostic Technology

Active case finding of AMR in humans, animals, and food chain

NegativesPositives

Confirm disease absence

Confirm disease presence

Intervention: treatment, therapy, etc.

Phenotypic response –Promotes just-in-time (JIT) use of antibiotics

Early diagnosis early response early recovery healthy population

Core Technologies

PCR-less genomic DNA detection

E. coli DNA

Electrochemical detection

Triage technology:MNP-F# for rapid screening

Diagnostic technology:AuNPs for genomic detection

Triage Technology

Active case finding of AMR in humans, animals, and food chain

NegativesPositives

Confirm disease absence

Confirm disease presence

Intervention: treatment, therapy, etc.

Matting: Microbial load is higher than normal

Triage

Diagnose

Monitor

• Magnetic nanoparticles (MNP) are coated with functional groups (F#) reactive to bacterial surfaces.

• F# acts as a pattern recognition receptor to distinguish pathogen-associated molecular patterns

(LPS, mannose, peptidoglycans, lipoproteins, etc.)

• Magnetic attraction is weak enough that van der Waals forces of F# are sufficient to prevent magnetic clumping or agglomeration.

• Nanoscale size (180-500 nm) results in colloidal suspension through Brownian motion.

Core Technology 1: Functionalized MNP

MNP powder

MNP in colloidal form suspension.

TEM image of MNP in solution; positively charged zeta potential

+

+

+++

+

Core Technology 2: AuNP-probe for PCR-less DNA Detection

TEM images of AuNPs, ~15 nm in diameter. Well-dispersed, uniform size and shape.

Alocilja AuNP solution during synthesis.

Positive Negative

Colorimetric detection takes < 60 minRequires only a heating block

Bacterial DNA Detection Using DNAprobe-Gold Nanoparticles (AuNP)

+Reactant

CT1: MNP Extraction & Matting

Features:

• Simple: 3-step process

• Affordable: < $0.05/test

• Rapid: < 10 min

• Equipment-free (uses a simple magnet)

• No electricity, No refrigeration

• No antibodies or protein

• Long shelf-life at room temperature

• High throughput

Procedure

1. Add sample (e.g. urine) to MNP tube

2. Magnetically separate for 1 minute

3. Dispose liquid and analyze MNP mat

1 2 3

Triage

Diagnose

Monitor

Proposed Mechanism of F#-Cell Interaction

• MNP in solution increases particle density and surface

area and thus increases Brownian motion of bacteria and

hydrodynamic interaction between bacteria and MNP

• Ionic/electrostatic interaction between F# (+ charge) with

bacterial cell surface (- charge)

• Carbohydrate-binding proteins on bacterial cell wall with

F#

•

• All forces taken together provide a strong cell-F# binding

Source of some images: Google images

Carbohydrate groups on MNP surface

++ ++

++++

+Cell

Proposed Mechanism for Surface Matting

• Hydrophobic/hydrophilic interaction between cell wall and tube

surface

• Hydrophobicity of cells can increase the propensity of

microorganisms to adhesion on biotic and abiotic surfaces.

• Hydrophobic cells (non-polar) adhere more strongly to hydrophobic

surfaces, while hydrophilic cells (polar) strongly adhere to

hydrophilic surfaces.

• Mycobacterium sp. have hydrophobic envelopes and show co-

aggregation.

High contact angle Low contact angle

cellcell

Tube surfaceTube surface

Healthy urineE. coli- infected

urine

Triage Technology

Active case finding of AMR in humans, animals, and food chain

NegativesPositives

Confirm disease absence

Confirm disease presence

Intervention: treatment, therapy, etc.

Step 2: Diagnostic DNA biosensor - to determine specific disease

Step 3: Monitor treatment efficacy by matting – determines AMR case

Step 1: Triage by Matting Using MNP-F#

Triage technology:MNP-F# for rapid screening

Principle: Matting will show when microbial load is higher than normal.

Extraction & Concentration by MNP for RT-PCR

MNP extraction of microbial load directly from blood (EDTA or BD Bactec tube)

Advantages of MNP over filter method in blood samples:• Quick extraction and concentration • Multi-processing: 96-well plates with magnetic blocks • Cheap: $0.05/test for MNP vs $1/test for syringe filter

Step 1

Add blood sample to

MNP tube, shake, let

stand 5 min

Step 2

Place tube in

magnetic

separator,

remove

supernatant

Confirm

Extraction

Re-suspend

MNP-cell in PBS

and lyse cells on

heating block

Confirm

Extraction

Use extracted

genomic DNA

from supernatant

into PCR assay

Results

Experiments done in 14 pathogens including S. aureus (LDH1 gene) and MRSA (mecAgene). Data from MSU.

MNP-F# For TB Extraction

Triage

M. smegmatis grown in agar. Morphology is

wrinkled, creamy yellow after a long period

of growth.

M. smegmatis grown in agar previously extracted by

MNP. Note the brown MNP surrounding the bacteria.

Table 1. Capture efficiency of MNP for M. smegmatis extraction in artificial sputum

Range of cell count Ave cell count Capture Efficiency No. tests

10,000

Lowest detection of

current bacilloscopy

106-240 10^2 90% 12

24-47 10^1 91% 15

2-9 10^0 91% 21

Lowest detection of our

biosensor

MNP-F# For TB Extraction

MNP-F# for Matting in Clinical Fluids

Healthy urine (N) and

bacteria-infected urine (P).

Clinical data from KU,

Nepal.

MNP matting

from rheum fluid.

Clinical data

from UCV, Peru. Image from the web.

Matting of tracheal swabs from 16 backyard chicken sick with the New Castle

disease (confirmed using rapid antigen detection kit). Rightmost tube is control.

Data from AFU, Nepal.

Matting of 4 liver samples from 4 dead chicken (from one flock); 3 confirmed of

Colibacillosis by culture & biochemical test (took 3 days to confirm)

+ - + + Con

Matting in Chicken Samples

Swabs taken from various body parts of a diseased chicken (L

to R): cecal, ovary, liver 1, liver 2, cloaca, trachea 1, trachea 2.

Matting in Chicken Samples

Tracheal swabs from healthy (D)

and diseased chicken (L & K).

Quickly monitor disease distribution in body parts.

Blank 1 2 3 4

1 2 3 4 Blank

Pre-onset Detection

105 CFU/mL

E. coli infection

User InterfaceMNP mat density and area could be compared

to a visual reference, similar to pH paper.

A smartphone app could be used to

scan the image and display

concentration.

Alocilja Research Group

www.egr.msu.edu/alocilja

alocilja@msu.edu

vasherma@msu.edu

Cellphone Enabled Processing

Saliva samples 1-4 and Blank (PBS). The next day,

sample donor #2 had fever, cough and cold. Matting

was able to predict the health condition of #2 prior

to getting sick. USA.

Disease Monitoring Using Saliva

Quantitative Black Pixel Count

1 2 3 4 Blank

Adjust threshold for matting

Disease Threshold With Control

Triage Technology

Active case finding of AMR in humans, animals, and food chain

NegativesPositives

Confirm disease absence

Confirm disease presence

Intervention: treatment, therapy, etc.

Step 2: Diagnostic DNA biosensor - to determine specific disease

Step 1: Triage matting - to determine sick/not sick

Step 3: Monitor treatment efficacy by matting – determines AMR case

Step 2: Diagnostic Technologies

Triage

Diagnose PCR-less genomic DNA detection

E. coli DNA

Electrochemical detection

Core Technology 2: AuNPs for genomic detection

Fluorescence image

of the TB positive

sputum. Microscopist

graded the sample

3+; without the MNP,

sample was graded

2+.

Triage and Confirmation

Diagnostic test

Triage Assay

TB negative sputum.

Clinical data from India.TB positive sputum.

Clinical data from India.

Sputum sample

Smear microscopy with

Ziel-Neelsen staining.

PCR-less genomic DNA detection

Conventional AFB Smear Microscopy – with Ziehl-Neelsen Staining

MNP-assisted AFB Smear Microscopy – with Ziehl-Neelsen Staining

Detection: MNP-Assisted Smear Microscopy

Place sputum on slide

and stain Stained M. Tuberculosis using

conventional Acid Fast Bacilli (AFB)

smear microscopy (red bacilli in center).Processing time: 20 min 10 min = 30 min

Step 1

Add sputum to MNP

tube, shake, let stand

5 min

Step 2

Place tube on

magnetic separator,

remove supernatant

Step 3

Observe matting and

transfer sample on

slide to stain

1`

Stained M. Tuberculosis extracted

by MNP followed by Acid Fast

Bacilli (AFB) smear microscopy

(red bacilli in center).

Steps 1-3:

10 min

DNA detection

Viral RNA Detection using DNAzyme-Gold Nanoparticles (AuNP)

TEM images of AuNPs, ~15 nm in diameter. Well-dispersed, uniform size and shape.

Gold NP solution during synthesis.

Dengue RNA extracted from Dengue-

carrying mosquitoes. Data from the

Philippines.

Reagents do not need refrigeration; transportable; RNA extraction by grinding.

Positive Negative

+ Salt

Target RNA Non-target RNA

+ +Probe-AuNP

Quantitative analysis of the

colorimetric detection of DENV-3

using UV/Vis spectrophotometry.

Data from the Philippines.

Step 2: Genomic Detection Using Gold Nanoparticles (AuNP)

Step 3: Monitor by Matting

Triage Technology

Active case finding of AMR in humans, animals, and food chain

NegativesPositives

Confirm disease absence

Confirm disease presence

Intervention: treatment, therapy, etc.

Step 2: Diagnostic DNA biosensor - to determine specific disease

Step 1: Triage matting - to determine sick/not sick

Step 3: Monitor treatment efficacy by matting – determines AMR case

A. Biosensor Technology

Triage

Diagnose

Monitor

Dose-response in Dengue virus detection

0 µg/mL35 µg/mL

Diluted by a factor of 2

Reproducibility test for Dengue virus detection. Potential profile of

antibiotic resistance.

Step 3: Monitor Treatment Efficiacy by Matting

Figure 1. On Monday, 12/12, saliva of sick patient (cold and cough with slight fever) was taken and tested (day 0). On

Tuesday, 12/13, patient was given antibiotic to be taken over 10 days. Starting on Wednesday, saliva of patient was

monitored everyday thereafter (day 1-10). Figure 1 shows that AMN matting was getting thinner as patient progresses to

recovery. Patient is showing full recovery on 8th day.

0 1 2 3 4 5 6 7 8 9 10 Control

Figure 2. AMN matting

before antibiotic

treatment (left) and end

of antibiotic treatment

(right). Black pixel count of matting in Fig 2.

Control 0 10Control 0 1 2 3 4 6 7 8 9 10

Black pixel count of matting in Fig 1.

Monitor by Matting for Treatment Efficacy

Disease threshold in matting with control

Control 0 1 2 3 4 6 7 8 9 10

1 2 3 4 Blank 0 1 2 3 4 5 6 7 8 9 10 Control

Blank 1 2 3 4

Disease Threshold With Control

Global Alliance for Rapid Diagnostics (GARD)

• GARD follows the peer-to-peer (P2P) network architecture where each node represents a member.

• Virtual community

• Research tasks and workloads on diseases to monitor are distributed among peers according to their national interests and commitments.

• Decentralized, equipotency of participants, free cooperation of equals to achieve the same goals.

Fundamental Guidelines: • Peer production – collaborators participate in generating

data for the benefit of society

• Peer property – although individual authorship is recognized, the use-value of data and procedures are freely accessible to the members.

Global Biosurveillance Network

Uber-like consortium structure

Membership: 160 members from 14 countries

GARD Output:

• Joint conferences - 2

• Faculty trainings – 10

• Student exchanges - 2

• Sponsor short courses - 1

• Joint peer-reviewed journal publications - 4

• Data from various nodes form the biosurveillance backbone• Data will be mapped in real time to assist in determining:

- initiation and occurrence of infections and potential epidemics- direction of infection flow- initiation, degree, & spread of antimicrobial resistance - AMR in the food chain- relationship of human-animal-wildlife interactions

Global Biosurveillance Network

Data from triage & biosensor

Schematic of the biosurveillance map at the community level

Schematic of the biosurveillance map at the global level

Environmental data & other data sources

Issue alerts

Prioritize resources

Implement control programs

Actionable outcomes community level

Syndromic data

Issue alerts

Prioritize resources

Implement control programs

Actionable outcomes global level

Data from global partners

Active Case Finding from Various Points

Within communities:

• Emergency rooms

• Human and animal clinics

Supply chain:

• Antibiotic prescription

• Antibiotic use in animal feed

• Prices of antibiotics

• Availability of antibiotics

• Other uses of antibiotics

• Meat product export

• AMR in soils and water

Across countries through partnerships:

• Rural settings

• High population

• High animal production

Summary of Results

Highlight of Results:1. Triage nano-biosensor (TNB) provides quick phenotypic screening:

- Simple to use: 3-step process- Affordable and accessible: < $0.05/test- Rapid: < 10 min- Antimicrobial susceptibility- Actionable outcomes: guides antibiotic use, allocates resources

2. PCR-less DNA biosensor provides specific test- Specific- Affordable: < $2/test- Rapid: ~2 h (from lysis to detection)

3. Data integration- Phenotypic, genomic, syndromic, historical data- Global biosurveillance of antimicrobial distribution

Research Group:

• Graduate students

• Postdocs

• Professorial assistants

• High school interns

• Co-investigators

• Collaborators

For a complete listing, refer to:

http://www.egr.msu.edu/alocilja/

Acknowledgment of Funding & Research Group

Thank you for your attention!

Funding:

• W.K. Kellogg Foundation

• Grand Challenge Canada

• Midland Research Institute

for Value Chain Creation

• USAID