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The Amazing Race: Pathogen Detection and Quantification

The Amazing Race: Pathogen Detection and Quantification

Each winter, the flu season peaks in January as new strains of virus emerge and spread among school age children, elderly and immunocompromised members of the population.

Diagnosis of flu, and other infectious diseases, puts serious strain on public health labs.  But the intense pressure to handle more samples faster is driving development of new innovative technologies for detection and treatment of all kinds of infectious diseases.

This past month I had the opportunity to attend a section of talks at the CHI Next Generation Dx Summit in Washington D.C. focusing on two new methods that streamline detection and quantification of microbial pathogens (and antibiotic resistance) in high throughput. These new technologies promised the ability to gather far more information from a single patient sample at the same time than was ever before possible and in speeds not currently achievable with conventional methods.

Speed is key

A constant issue raised throughout the Dx Summit was the need for faster, easier, and multiplex methods for processing patient samples especially during outbreaks.

Laboratories have the need to detect increasing numbers of variants and strains of viruses or bacteria from patient samples along with handling increasing numbers of samples due to panic in the public when outbreaks occur.

Suddenly, doctors and emergency rooms become unindated with people who fear they might have the current variant of flu and want to be tested.  With news spreading as fast as viruses, let’s see what technologies two companies have developed to address this issue.

Method 1: Combined PCR and Capillary Electrophoresis

Capillary electrophoresis (CEP) is a well established method for detecting PCR products based on size separation. In order to turn this into a high throughput real-time detection and quantification platform, PrimeraDx combined, for the first time, CEP with PCR to create the ICEPlex system. A very simple sample-to-answer machine.

The benefit of this approach is that it allows for multiplexing of up to 60 targets per patient sample and detection in real-time.

After the amplification cycle, samples are shifted inside the instrument to a CEP deck where aliquots are removed and measured to determine the presence and amount of virus. The assay requires primer sets for each organism (a flu assay would have variants of flu strains or other respiratory viruses) with one primer fluorescently labeled for detection on capillary electrophoresis system.

Where real-time PCR is limited to 4-6 targets per assay due to limitations in the number of filters that can detect colors, the ICEPlex’s STAR assay can be expanded to a wide range of targets because it is not discriminating based on color.

And because the detection is occurring in real-time, pathogen load can be quantitatively determined. All assays come with standards and internal controls.

Method 2: Combined real-time PCR and Microarray

An alternative approach to high throughput multiplex analysis comes from Eppendorf.

Their groundbreaking system, called Real-time Array PCR (RAP), use PCR as a front end but finishes with a microarray for highly multiplexed detection and quantification.

Eppendorf’s presentation at the conference focused on the causes of noscomial infections that lead to ventilator associated pneumonia (VAP) and the 375,000 deaths in the US and Europe each year.

Because this disease can be caused by many multiple drug resistant species of bacteria, they used their system to develop a test for 15 primary pathogens involved in VAP and 16 drug resistance markers (plus 1 control), to enable rapid and accurate treatment of patients with correct antibiotics.

Traditional methods for detection of this disease require 3 days of culturing to diagnose VAP (and hence a delay in starting the correct antibiotic regime) while the RAP method can provide the results in just 3 hours.

The key is a novel enzyme used for PCR that functions under high salt conditions, called SuperSaltTM Taq Polymerase, which allows for hybridization to the microarray to occur in the same buffer as amplification.

PCR products are directly hybridized to the solid support, elongated, and detected in real-time. The RAP system is a closed instrument that uses centrifugation for the transport of liquids within the chip allowing for simple set up of the reaction and hands free results. The RAP method revolves around 12 patent families including proprietary primer and probe design which requires use of their software for multiplex assay development.

Infectious disease drives innovation

Both of these methods merge existing technologies in a new way to allow for rapid and sensitive detection of pathogens with minimal handling.  It is clear that as public awareness of infectious diseases increases, so does our need to respond quickly when outbreaks occur.

New technologies designed to reduce time to diagnose and treat patients will be integral to reducing the ability of infectious disease to spread and cause public alarm.

So which of these two new platforms do you think sounds more promising  for fast multiplex analysis of infectious disease organisms?

1 Comment

  1. Roberto on August 28, 2009 at 4:37 pm

    Very interesting technologies, thank you!
    In these times of health reforms, I wonder how all this will stand the hurricane. I’m not from USA so I won’t enter the (extremely complex) political debate, but I’m sure that if access to medicine has to get broader and cheaper, there’ll be much less room for diagnostic “fireworks shows” like these ones… We’ll see!

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