Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from gene-quantification.de

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.

Lab-in-a-Box 

In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab

Lab-on-a-Robot

Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.

Lab-on-a-Drone

A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from tamu.edu

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Frightening Fungus Among Us

  • Clinical Alert for Candida auris (C. auris) Issued by CDC
  • US Concerned About C. auris Misidentification and Drug Resistance
  • Sequencing C. auris DNA in Clinical Samples is Preferred for Identification
Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

When I was a kid and didn’t know better, there was a supposedly funny rhyme that “there’s fungus among us.” While this saying is thankfully passé nowadays, the growing number of infections by a formerly obscure but deadly fungus is frightening. This so-called “superbug” is an antibiotic-resistant fungus called Candida auris (C. auris) that’s worth knowing about, and is the fungal focus of this blog.

First, Some Fungus Facts

Fungi are so distinct from plants and animals that they were allotted a biological ‘kingdom’ of their own in classification of life on earth, although that was only relatively recently, i.e. 1969. There are 99,000 know fungi, which exist in a wide diversity of sizes, shapes and complexity that extends from relatively simple unicellular microorganisms, such as yeasts and molds, to much more complex multicellular fungi, such as mushrooms and truffles.

It was previously thought that genomes of all fungi are derived from the genome of the model fungus Saccharomyces cerevisae, which has been used in winemaking, baking and brewing since ancient times. However, genome sequencing of more than 170 fungal species has revealed that, while the genome size of S. cerevisae is only ~12 Mb, seven species of fungus have genome sizes larger than 100 Mb. This is attributed to various evolutionary pressure-factors generating transposable elements, short sequence repeats, microsatellites, and genome duplication, and noncoding DNA.

Fungal cell walls are made up of intertwined fibers mostly comprised of long chains of chitosan, the same tough compound found in the exoskeletons of animals such as spiders, beetles and lobsters. The chitin in fungal cells is entangled with glucans and other wall components, such as proteins, forming a mass that protects the cell membrane behind it—and posing a formidable barrier against antifungal drugs.

Taken from Wikipedia.org

Taken from Wikipedia.org

In researching whether there are any nucleic acid drugs against fungi, I found one early patent by Isis (now Ionis) Pharmaceuticals for use of antisense phosphorothioate-modified oligonucleotides for the treatment of Candida infections, but virtually no other reports. I suspect that will change in the future as pathogenic fungi and other disease-causing microbes become more resistant to conventional drugs.

Fungal infections of the skin are very common and include athlete’s foot, jock itch, ringworm, and yeast infections. While these can usually be readily treated, infections caused by pathogenic fungi have reportedly risen drastically over the past few decades. Moreover, with the increase in the number of immunocompromised (burn, organ transplant, chemotherapy, HIV) patients, fungal infections have led to alarming mortality rates due to ever increasing phenomenon of multidrug resistance.

Segue to a Serious Situation

Emergence of drug-resistant fungi is, in part, the segue to the serious story of the present blog. The other part being incorrect identification of a certain fungus as being a common candida yeast, which is not only scary but seemingly inexcusable in today’s era of highly accurate PCR-based assays to accurately identify microorganisms. Here’s the situation in a nutshell.

  1. auris infection, which is associated with high mortality and is often resistant to multiple antifungal drugs, was first described in 2009 in Japan but has since been reported in countries throughout the world. Unlike many Candida infections, C auris is a hospital-acquired infection that is contracted from the environment or staff of a healthcare facility, and it can spread very quickly.

To determine whether C. auris is present in the United States and to prepare for the possibility of transmission, the Centers for Disease Control (CDC) and Prevention issued a clinical alert in June 2016 requesting that C. auris cases be reported.

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

This official alarm bell, if you will, was triggered by the following facts:

  • Many isolates are resistant to all three major classes of antifungal medications, a feature not found in other clinically relevant Candida
  • auris identification requires specialized methods such as a MALDI-TOF mass spectrometry or sequencing the 28s ribosomal DNA, as pictured below.
  • Using common methods, auris is often misidentified as other yeasts, which could lead to inappropriate treatments.

The CDC subsequently found that seven cases were identified in Illinois, Maryland, New York and New Jersey. Five of seven isolates were either misidentified initially as C. haemulonii or not identified beyond being Candida. Five of seven isolates were resistant to fluconazole; one of these isolates was resistant to amphotericin B, and another isolate was resistant to echinocandins. While no isolate was resistant to all three classes of antifungal medications, emergence of a new strain of C. auris that is would pose a serious public health issue.

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Based on currently available information, the CDC concluded that these cases of C. auris were acquired in the U.S., and several findings suggest that transmission occurred:

  • First, whole-genome sequencing results demonstrate that isolates from patients admitted to the same hospital in New Jersey were nearly identical, as were isolates from patients admitted to the same Illinois hospital.
  • Second, patients were colonized with auris on their skin and other body sites weeks to months after their initial infection, which could present opportunities for contamination of the health care environment.
  • Third, auris was isolated from samples taken from multiple surfaces in one patient’s health care environment, which further suggests that spread within health care settings is possible.

A related Fox News story adds that C. auris was found on a patient’s mattress, bedside table, bed rail, chair, and windowsill. Yikes!

While the above situation in the U.S. might not seem particularly worrisome to you, the potential for emergence of more infectious C. auris strains with higher lethality should be of concern. That has already reportedly occurred in several Asian countries and South Africa. Obviously, deployment of the best available methods for pathogen identification can, in principle, lessen the likelihood of the emergence and/or spread of C. auris in the U.S. and other countries.

Case for Point-of-Care C. auris Nanopore Sequencing?

Taken from extremtech.com 

Taken from extremtech.com

Regular readers of my previous blogs know that I’m an enthusiastic fan of the Oxford Nanopore Technologies minION sequencer, which is proving to be quite useful for characterizing pathogens in very remote regions on Earth—and even on the International Space Station to diagnose astronaut infections! Notwithstanding various current limitations for minION sequencing of microbes, it seems to me that it would be relatively straightforward to generate minION data for many available samples of pathogenic fungi and genetically related microbes to assess the feasibility using minION for faster, cheaper, better unambiguous identification of C. auris minION in centralized or Point-of-Care applications.

Taken from rnaseq.com

Taken from rnaseq.com

If you think this suggestion is farfetched, think again, after checking out these 2016 publications using minION:

The 51.4-Mb genome sequence of Calonectria pseudonaviculata for fungal plant pathogen diagnosis was obtain using minION.

The first report of the ~54 Mb eukaryotic genome sequence of Rhizoctonia solani, an important pathogenic fungal species of maize, was derived using minION.

Sequence data is generated in ~3.5 hours, and bacteria, viruses and fungi present in the sample of marijuana are classified to subspecies and strain level in a quantitative manner, without prior knowledge of the sample composition.

CDC on C. auris Status and FAQs

In the interest of concluding this blog with the most up-to-date and authoritative information, I consulted the CDC website and found statements and replies to FAQs that are well worth reading at this link.

As a scientist, my overriding question concerns the lack of adoption of improved microbiological methods by hospitals and clinics. The above noted misidentifications of C. auris infections resulting from use of flawed lab analyses seems unacceptable. Although I don’t know all the facts or statistics to generalize, I suspect that there are other incorrect lab analyses due to use of outdated methods. On the other hand, I’m hopeful that, with the FDA’s widely touted Strategic Plan for Moving Regulatory Science into the 21st Century, the section entitled Ensure FDA Readiness to Evaluate Innovative Emerging Technologies—think nanopore sequencing—becomes actionable, sooner rather than later.

Changing established—dare I say entrenched—clinical lab tests is not simple or easy, but if it doesn’t begin it won’t happen, about which I’m quite certain. I can only wonder why development of infectious disease analytical methods and treatments seem to require a crisis. Sadly, I think it boils down to the complexities and socio-political dynamics of who pays.

Frankly, it’s my personal opinion that maybe it’s time Thomas Jefferson’s philosophy about hammering guns into plows is directed to health care.

Postscript

After writing this blog, I learned that T2 Biosystems has received FDA approval to market in the U.S. the first direct blood test for detection of five yeast pathogens that cause bloodstream infections: Candida albicans and/or Candida tropicalis, Candida parapsilosis, Candida glabrata and/or Candida krusei.

Yeast bloodstream infections are a type of fungal infection that can lead to severe complications and even death if not treated rapidly. Traditional methods of detecting yeast pathogens in the bloodstream can require up to six days, and even more time to identify the specific type of yeast present. The T2Candida Panel and T2Dx Instrument (T2Candida) can identify these five common yeast pathogens from a single blood specimen within 3-5 hours.

T2Candida incorporates technologies that break the yeast cells apart, releasing the DNA for PCR amplification for detection by greatly simplified, miniaturized nuclear magnetic resonance (NMR) technology, as can be seen in this video.

In my opinion, this fascinating new technology is another example of what could be rapidly deployed toward detecting C. auris.

Update on Zika Virus Detection by RT-PCR

  • Various RT-PCR Assays for Zika Virus Have now Received Emergency Use Authorization (EUA) from the FDA
  • Quest Diagnostics’ Assay Approved by FDA for General Use as a Zika Test
  • Troubled Theranos Touts New “miniLab” for Zika EUA
  • Vaccine Development Progressing Albeit Relatively Slowly
Aedes aegypti mosquito. Taken from wcvb.com

Aedes aegypti mosquito. Taken from wcvb.com

In January 2016, I posted a blog about the then emerging public awareness of Zika virus (ZIKV), which is spread by the bite of infected mosquitos—primarily the Aedes aegypti mosquito. Sadly, ZIKV can be passed from a ZIKV-infected pregnant woman to her fetus leading to development of a brain defect (microcephaly) and/or other malformities. Moreover, ZIKV is now associated with sexual transmission and blood transfusion. This is scary news.

This update was prompted by the additional fact that ZIKV is on the rise and there is still no vaccine, according to the Centers for Disease Control and Prevention (CDC)—my “go to” source for loads of authoritative information about this infectious disease. Weekly updated ZIKV-infection “case counts” in US States and US Territories were given as ~1,200 and ~6,500, respectively on Aug 10th, and increasing to ~3,400 and ~20,000 on Sep 21st —only 6 weeks later!

As I pointed out in my previous blog on ZIKV, absent an anti-ZIKV vaccine, there is considerable interest in mosquito abatement as well as early detection, notably by reverse-transcription PCR (RT-PCR) of this RNA Flavivirus. Now that Zika infection by mosquitos in Florida has been found, leading to several CDC warnings, I thought it would be both apropos and technically interesting to provide the following update on ZIKV structure and RT-PCR.

ZIKV Genome and Structure

As of last month, my PubMed search of “Zika [Title/Abstract] AND RT-PCR [Anywhere]” gave 55 publications. Incidentally, there are now a number of Zika genome sequencing publications, such as this lead reference. It’s worth noting that ZIKV genome sequencing enables monitoring the potential evolution of new genomic variants that might foil existing RT-PCR assays and guide the selection of new primers for RT-PCR.

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

It’s also worth pointing out that Zika’s overall molecular capsid structure is enveloped, spherical, and has a diameter of ~40 nm in diameter, as depicted below in schematic form, and pictured in the accompanying electron microscopic image. The surface proteins are arranged in an icosahedral-like symmetry.

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

ZIKV RT-PCR

In the interest of giving explicit credit to various investigative groups who have developed RT-PCR assays for ZIKV, here are links and short snippets I’ve selected from publications, in chronological order, found in the aforementioned PubMed search. Interested readers are encouraged to check out the original publication for details.

The first report of an RT-PCR assay for ZIKV appears to be by Faye et al. in 2008 at the Institut Pasteur de Dakar in Senegal, who targeted the envelope protein coding region and tested ZIKV isolates previously collected over a 40-year period from various African countries and hosts. The assay’s detection limit and repeatability were respectively 7.7pfu/reaction and 100% in serum; none of 19 other Flaviviruses tested were detected. Faye et al. in 2013 extended this work to include quantitative RT-PCR detection of ZIKV and evaluation with samples from field-caught mosquitoes.

Kinetics of ZIKV detection in urine compared to serum from 6 patients was described by Gourinat et al. in 2015 at the Institut Pasteur, Noumea, New Caledonia using RT-PCR primers and probes previously reported by others. Urine samples were positive for ZIKV more than 10 days after onset of disease, which was a notably longer period than 2-3 days for serum samples. These researchers concluded that urine samples are useful for diagnosis of ZIKV infections, and are preferred to serum wherein virus titer diminishes more rapidly.

Musso et al. in 2015 working in Tahiti, French Polynesia with 1,067 samples collected from 855 patients presenting symptoms of Zika fever found that analysis of saliva samples increased the rate of detection of ZIKV at the acute phase of the disease compared to serum samples. They noted that saliva was of particular interest when blood was difficult to collect, especially for children and neonates.

Most recently, Xu et al. in 2016 in China reported the development of a SYBR Green (intercalator dye)-based qRT-PCR assay for detection of ZIKV. Although their results indicate that the assay is specific, it’s important to note that SYBR-type detection can be subject to nonspecific artifacts, for which TriLink’s proprietary CleanAmp™ Primers can be investigated to potentially ameliorate such problems, as discussed in this downloadable pdf publication by TriLink researchers.

ZIKV In Vitro Diagnostic Assays

As detailed elsewhere, the Secretary of Health and Human Services (HHS) earlier this year determined that “there is a significant potential for a public health emergency that has a significant potential to affect national security or the health and security of United States citizens living abroad and that involves Zika virus.” The Secretary of HHS further declared that “circumstances exist justifying the authorization of the emergency use of in vitro diagnostics for detection of Zika virus…”.

The following RNA-based assays and suppliers are currently listed by the FDA for this Emergency Use Authorization (EUA):

  • xMAP® MultiFLEX™ Zika RNA Assay (Luminex Corporation)
  • VERSANT® Zika RNA 1.0 Assay (kPCR) Kit (Siemens Healthcare Diagnostics Inc.)
  • Zika Virus Real-time RT-PCR Test (Viracor-IBT Laboratories, Inc.)
  • Aptima® Zika Virus Assay (Hologic, Inc.)
  • RealStar® Zika Virus RT-PCR Kit U.S. (Altona Diagnostics)
  • Zika Virus RNA Qualitative Real-Time RT-PCR (Focus Diagnostics)
  • Trioplex Real-time RT-PCR Assay (CDC)

Among these, two are particularly notable—in my opinion. Trioplex Real-time RT-PCR Assay is a multiplexed laboratory test developed by the CDC to simultaneously detect ZIKA, dengue virus, and chikungunya virus RNA, each of which can be transmitted primarily by Aedes aegypti mosquitos to cause infections with similar symptoms. Consequently, it is useful to have a single test that can detect each of these viruses in the same sample. Full details for the Trioplex assay are provided in a 40-page downloadable pdf from the CDC at this link.

In brief, this CDC-developed test uses virus-specific primer pairs and fluorogenic hydrolysis probes each differentially dual-labeled with fluorescent reporter and quencher dyes for in vitro detection of complementary DNA (cDNA). The detection follows reverse-transcription of RNA isolated from clinical specimens including serum, cerebral spinal fluid, urine, and amniotic fluid. It’s worth pointing out that I subsequently found a publication in 2016 by several collaborating academic groups regarding an analogous triplex RT-PCR assay for the same three viruses.

The other notable assay is Zika Virus RNA Qualitative Real-Time RT-PCR developed by Focus Diagnostics, which in April 2016 was the first of the aforementioned ZIKV tests to receive authorization by the FDA for use by qualified labs to detect ZIKV RNA in blood samples of those meeting CDC clinical criteria or of people who may have lived in or traveled to an affected location or had other exposure to the virus. Quest Diagnostics, the parent company of Focus Diagnostics, announced it would make the test broadly available to physicians, including those in Puerto Rico in May 2016.

ZIKV EUA Sought by Theranos for Its new “miniLab”

Theranos, which has been in the news regarding troubles over its stealthy proprietary system for finger-stick blood tests, appears to be pivoting its strategic plans. It announced at the August 2016 American Association of Clinical Chemistry Meeting its R&D for a new, fully automated miniLab system, including analytical and method comparison results of its ZIKV nucleic acid-amplification-based assay.

According to its press release, the company collected finger-stick samples from subjects, including people in the ZIKV-infested Dominican Republic, and shipped those to Palo Alto, California to run on the miniLab. Although I was unable to obtain these particular results, I did find the following figure at the TechCrunch website showing functional components of the miniLab along with an article by Sarah Buhr that’s worth a quick read, in my opinion.

Taken from techcruch.com

Taken from techcruch.com

According to the aforementioned Theranos press release, the company has submitted assay validation data for this Zika assay to the FDA for an EUA. The company also states that it is unaware of any currently available capillary (i.e. finger-stick) test for ZIKV.

I’ll stay tuned for future general information about the miniLab, as well as information specifically related to ZIKV. If I hear of anything, I’ll add as a comment here or in a new post with technical details about its nucleic acid assays.

ZIKV Vaccine Status

I hope this blog has convinced you that RT-PCR of ZIKV has provided improved molecular diagnostics. I’m guessing, that like me, you find it unfortunate that there isn’t a proven anti-ZIKA vaccine as of yet. This is especially frustrating given the fact that Zika disease has been known for more than 50 years, and that it is evidently on the rise globally, including in the USA according to regularly updated CDC statistics.

In February 2016, the Obama administration requested $1.9 billion in funding for the NIH to develop a ZIKV vaccine. The US Congress continues to be deadlocked by partisan politics despite the fact that Florida state and local officials are scrambling to contain the ZIKV outbreak in Miami Beach. This outbreak poses a serious threat to the health of residents, as well to visitors who drive the region’s $24 billion-a-year tourism industry.

Nevertheless, some progress has been reported for US studies in monkeys, and US-based Inovio says it’s received FDA approval to begin studies in humans. Outside the US, Sanofi Pasteur in France is said to be poised for initiating its trials in humans, and French biotech company Valneva is reported to have succeeded in generating a ‘highly purified inactivated vaccine candidate’ using the same technical approach it used for its encephalitis vaccine that is marketed in the United States and Europe.

Lagging—dare I say “glacially slow”—action against ZIKV by the World Health Organization is quite disappointing to me, and is reminiscent of what I’ve commented on in an earlier blog concerning this bureaucracy’s ineffective response to the Ebola virus. If I had my druthers, TriLink’s previously announced engagement by Battelle in development of Ebola mRNA vaccine would somehow materialize for a Zika mRNA vaccine. In this regard, GlaxoSmithKline is reported to be preparing research studies alongside the NIH’s Vaccine Research Center to test a self-amplifying mRNA vaccine technology for Zika. Interested readers can check out this link to a fascinating PNAS publication by Geall et al. on biosynthetic self-amplifying mRNA vaccines delivered in lipid nanoparticles.

As always, your comments are welcomed.

Postscript

Taken from Fleming et al. (2016) ACS Infectious Diseases

Taken from Fleming et al. (2016) ACS Infectious Diseases

One of my previous posts featured recent elucidation of biologically functional G-quadraplexes in living cells. Consequently, it’s apropos to mention here that Fleming et al. at the University of Utah have just now published the first analysis of potential G-quadruplex sequences (PQS) in the RNA genome of ZIKV. As depicted in this artistic cartoon, several PQS were found, with the most stable located near the end of the 3’ untranslated region (3′-UTR). Importantly, these investigators propose a rationale for screening G-quadruplex-binding compounds as a completely new class of anti-ZIKV drug candidates. In my opinion, this is a great example of how basic biochemical research can lead to new strategies for much needed antiviral drugs.

RNA World Revisited

  • Scripps Researchers ‘Evolve’ an RNA-Amplifying RNA Polymerase 
  • It’s Used for First Ever All-RNA Amplification Called “riboPCR”
  • TriLink Reagent Plays a Role in this Remarkably Selective in Vitro Evolution Method 
Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from scripps.edu

Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from scripps.edu

Those of you who regularly read my blog will recall an earlier posting on “the RNA World,” which was envisioned by Prof. Walter Gilbert in the 1980s as a prebiotic place billions of years ago when life began without DNA. That post recommended reading more about this intriguing hypothesis by consulting a lengthy review by Prof. Gerald Joyce. Now, Prof. Joyce and postdoc David Horning have advanced the hypothesis one step further by reporting the first ever amplification of RNA by an in vitro-selected RNA polymerase, thus providing significant supportive evidence for the RNA World. Following are their key findings, which were enabled in part by a TriLink reagent—read on to find out which one and how!

In Vitro Evolution of an RNA Polymerase

Horning & Joyce designed an in vitro selection method to chemically “evolve” an RNA polymerase capable of copying a relatively long RNA template with relatively high fidelity. The double emphasis on “relatively” takes into account that the RNA World would have many millions of years to evolve functionally better RNA polymerases capable of copying increasingly longer RNA templates with increasingly higher fidelity.

As depicted below, they started with a synthetic, highly structured ribozyme (black) wherein random mutations were introduced throughout the molecule at a frequency of 10% per nucleotide position to generate a population of 1014 (100,000,000,000,000) distinct variants to initiate the in vitro evolution process. Step 1 involved 5’-5’ click-mediated 1,2,3-trazole (Ø) attachment of an 11-nt RNA primer (magenta) partially complementary to a synthetic 41-nt RNA template (brown) encoding an aptamer that binds guanosine triphosphate (GTP). In Steps 2 and 3, the primer hybridizes to template and is extended by polymerization of A, G, C and U triphosphates (cyan).

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Step 4 involves binding of aptameric structures to immobilized GTP (green), then photocleavage of the 1,2,3-triazole linkage in Step 5, followed by reverse transcription to cDNA and conventional PCR in Step 6 for transcription into ribozymes in Step 7. Twenty-four rounds of this evolution by selection were carried out, progressively increasing the stringency by increasing the length of RNA to be synthesized by decreasing the time allowed for polymerization. By the 24th round, the population could readily complete the GTP aptamer shown below. Subsequent cloning, sequencing and screening were then used to characterize the most active polymerase, which was designated “24-3.”

The TriLink “Connection”

2'-Azido-dUTP (aka 2'-azido-UTP)

2′-Azido-dUTP (aka 2′-azido-UTP)

The aforementioned in vitro evolution process actually involves tons of experimental details that interested readers will need to consult in the published paper, which is accompanied by an extensive Supporting Information section. In the latter, a subsection titled Primer Extension Reaction describes 3’ biotinylation of the template RNA strand (brown in above scheme) using TriLink “2’-azido-UTP” (more properly named 2’-azido-dUTP) and yeast poly(A) polymerase, followed by click connection of the RNA template’s 3’-terminal 2’-azido moiety to biotin-alkyne. This very clever functionalization of the RNA template strand allowed for subsequent capture of the double-stranded primer extension reaction products on streptavidin-coated beads, followed by elution of the desired nonbiotinylated strand for GTP aptamer selection (Step 4 above).

Properties of RNA Polymerase 24-3

Needless to say—but I will—enzymologists and RNA aficionados will undoubtedly be interested in musing over the kinetic and fidelity properties of RNA polymerase 24-3.

The rate of 24-3 polymerase catalyzed addition to a template-bound primer was measured using an 11-nt template that is cited extensively in the literature to evaluate various ribozymes. It was found that the average rate of primer extension by 24-3 is 1.2 nt/min, which is ∼100-fold faster than that of the starting ribozyme polymerase randomly mutagenized for in vitro selection.

The NTP incorporation fidelities of the starting and 24-3 ribozyme polymerases on this 11-nt test template, at comparable yields of product, are 96.6% and 92.0%, respectively. Horning & Joyce noted that the higher error rate of 24-3 is due primarily to an increased tendency for G•U wobble pairing.

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Other longer RNA templates having various base compositions or intramolecular structures were also studied, with the stated “final test of polymerase generality” being use of 24-3 to synthesize yeast phenylalanyl tRNA from a 15-nt primer (in red right). The authors humorously describe the results as follows:

“Despite the stable and complex structure of the template, full-length tRNA was obtained in 0.07% yield after 72 h. This RNA product is close to the limit of what can be achieved with the polymerase, but is likely the first time a tRNA molecule has been synthesized by a ribozyme since the end of the RNA world, nearly four billion years ago.”

Exponential Amplification of RNA

PCR is the most widely used method for amplifying nucleic acids, and involves repeated cycles of heat denaturation and primer extension. The 24-3 RNA polymerase was used to carry out PCR-like amplification, but in an all-RNA system (named riboPCR by Horning & Joyce) using A, G, C, and U triphosphates and a 24-nt RNA template composed of two 10-nt primer-binding sites flanking the sequence AGAG. Somewhat special conditions were employed:

  • The concentration of Mg2+ was reduced to minimize spontaneous RNA cleavage
  • PEG8000 was used as a “molecular crowding” agent to improve ribozyme activity at the reduced Mg2+ concentration
  • Tetrapropylammonium chloride was added to lower the melting temperature of the duplex RNA

Under these conditions, 1 nM of the 24-nt RNA template was driven through >40 repeated thermal cycles, resulting in 98 nM newly synthesized template and 106 nM of its complement, corresponding to 100-fold amplification. Sequencing of the amplified products revealed that the central AGAG sequence was largely preserved, albeit with a propensity to mutate the third position from A to G, reflecting the low barrier to wobble pairing.

Amplification of a 20-nt template (without the central insert) was monitored in real time, using FRET from fluorescently labeled primers, and input template concentrations ranging from 10 nM to 1 pM. The resulting amplification profiles shown in the paper are typical for real-time PCR, shifted by a constant number of cycles per log-change in starting template concentration. A plot of cycle-to-threshold vs. logarithm of template concentration, also shown in the paper, was linear across the entire range of dilutions indicating exponential amplification of the template RNA with a per-cycle amplification efficiency of 1.3-fold.

Implications for the Ancient RNA World

It would be an injustice to Horning & Joyce if I would try to paraphrase their concluding discussion of this investigation, so here is what they say:

The vestiges of the late RNA world appear to be shared by all extant life on Earth, most notably in the catalytic center of the ribosome, but most features of RNA-based life likely were lost in the Archaean era. Whatever forms of RNA life existed, they must have had the ability to replicate genetic information and express it as functional molecules. The 24-3 polymerase is the first known ribozyme that is able to amplify RNA and to synthesize complex functional RNAs. To achieve fully autonomous RNA replication, these two activities must be combined and further improved to provide a polymerase ribozyme that can replicate itself and other ribozymes of similar complexity. Such a system could, under appropriate conditions, be capable of self-sustained Darwinian evolution and would constitute a synthetic form of RNA life.

Applications for Today’s World of Biotechnology

The aforementioned report by Horning & Joyce has received wide acclaim in the scientific press and world-wide public media as supporting the existence of a prebiotic RNA World, billions of years ago, from which life on Earth evolved.

While the academic part of my brain, if you will, fully appreciates the significance of these new insights on “living” RNA eons ago, the technical applications part of my brain is more piqued by possible practical uses of all-RNA copying or all-RNA riboPCR.

I, for one, plan to muse over possible applications of such all-RNA systems in today’s world of biotechnology, and hope that you do too, and are willing to share any ideas as comments here.

Spartan Cube—The World’s Smallest Molecular Diagnostic Device

  • Records Are Meant to be Broken—Including Those for PCR Diagnostics
  • Spartan Bioscience Claims It’s Cube is World’s Smallest Mol Dx Device 
  • The Cube was Launched at the Recent AACC Clinical Lab Expo 

Prologue

[22]-annulene. Taken from Wikipedia.com

[22]-annulene. Taken from Wikipedia.com

In the spirit of the recent Olympic games, and as the saying goes—records are meant to be broken. When I was in grammar school, a big buzz in sports was who would be the first to break the 4-minute mile; answer: Roger Bannister in 3 minutes and 59.4 seconds in 1954—now 17 sec faster. In my college craze days, it was how many persons can fit into a Volkswagen Beetle; answer: I couldn’t find the first feat, but the current record is 20 crammed into an old style Beetle in 2010. Then during my graduate organic chemistry studies, there was interest in synthesizing increasingly larger annulenes—completely conjugated CnHn monocyclic hydrocarbons akin to benzene (n=6); answer: [22]-annulene with n=22 (see below) synthesized by F. Sondheimer. But I digress…

Our collective fascination with records—and beating them—also applies to all sorts of instruments for health-related sciences, such as the most powerful MRI imaging systems (currently from GE) or longest-DNA-sequencing system (currently from PacBio). Due to the seemingly endless utility of PCR, there is a continual stream of claims for the fastest PCR system (currently from BJS Biotechnologies) or—more to point herein—smallest PCR system.

To wit, regular readers of my blog will recall an April 2016 post titled World’s Smallest Real-Time PCR Device, which referred to a hand-held system reported by Ahrberg et al. for real-time, quantitative PCR (qPCR). That system is pictured below next to the original system commercialized by ABI in the 1990s that weighed 350 pounds and was 7 feet long!

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

It seems that PCR records fall as easily as those in the Olympics. Not even a year later, Ahrberg’s claim is being challenged by Canadian company Spartan Bioscience, which recently introduced its Cube device at the 2016 AACC Annual Scientific Meeting & Clinical Lab Expo. Following are some technical details that I thought were worth sharing.

Taken from businessinsider.com

Taken from businessinsider.com

Cube Facts

Given its amazingly small size of only 4 x 4 x 4 inches, there apparently has been some remarkable engineering achievements to be able to squeeze-in what’s needed for the rapid heating and cooling required for PCR thermal cycling. Ditto for the optics required to enable fluorescence detection. Like other relatively small devices intended for emerging Point-of-Care (POC) applications in a doctor’s office or clinic, there is wireless connectivity to a laptop that serves as the user interface for operation and data analysis, as well as a power source for the Cube via a USB cable.

You’re likely wondering by now how much the Cube will sell for. Unfortunately, I was not able to obtain a list price from Spartan’s CEO at this time, so we’ll all have to wait and see.  I’ll post the answer as a comment to this blog as soon as I find out the price.

Inside the Cube—Perhaps 

I actually don’t know exactly what’s inside the Cube, but some possibilities of what might be are as follows. I’ve based these educated guesses on a Spartan Bioscience patent (US pat. no. 8,945,880) by Paul Lem and others entitled Thermal cycling by positioning relative to fixed-temperature heat source. As depicted below, a hot block provides a heat source at a fixed temperature to thermally cycle PCR reaction vessels that can be precisely moved by a micrometer to and from the hot block in a repeated manner.

Taken from US patent no. 8,945,880

Taken from US patent no. 8,945,880

As for how fluorescence might be measured to monitor each PCR reaction in real-time, one possibility is depicted below. Basically, each reaction tube is proximate to excitation light from an LED, and has a slit at the bottom for emitted light that is collected and processed into a typical real-time PCR curve.

Taken from US patent no. 8,945,880

Taken from US patent no. 8,945,880

Before the Cube

Prior to launching the Cube, Spartan Bioscience has been selling an FDA-Cleared in vitro diagnostic product called Spartan RX, which is also relatively compact, and carries out fully automated—“cheek swab-to-result”—PCR analysis of certain Cytochrome P450 2C19 (CYP2C19) genotypes. Roughly 1-in-3 people carry CYP2C19 mutations that can impair metabolism of a wide variety of commonly used drugs. Consequently, these PCR-based results are a valuable aid to clinicians in determining strategies for therapeutics that are metabolized by the Cytochrome P450 2C19.

I was favorably impressed by the fact that this CYP2C19 assay qualifies for reimbursement from Medicare and most insurers, according to the company’s website, which adds that there is an ongoing 6,000-patient clinical trial entitled Tailored Antiplatelet Initiation to Lessen Outcomes due to Clopidogrel Resistance after Percutaneous Coronary Interventions (TAILOR PCI).

The Spartan RX cheek swab POC results have been recently compared to centralized genotyping with a TaqMan® allelic discrimination assay (Life Technologies) using qPCR and with the GenID® reverse dot-blot hybridization assay (Autoimmun Diagnostika GmbH). Published results indicate excellent agreement, and led to the following conclusions by the authors: “Compared to both laboratory-based genotyping assays, the POC assay is accurate and reliable, provides rapid results, can process single samples, is portable and more operator-friendly, however the tests are more expensive.”

I look forward to finding out more about the Cube and the PCR results it can obtain. I’ll post more information on my blog as it becomes available. As usual, your comments are welcomed.

Postscript

To me, there’s something visually intriguing about a cube, perhaps because it’s one of the so-called Platonic Solids, which have been known since antiquity and studied extensively by the ancient Greeks.

Taken from acs.org

Taken from acs.org

Platonic solids such as the cube have also fascinated chemists, as evidenced by there being a substantial amount of published literature on the synthesis and physical properties of platonic solids. For example, cubane (C8H8) is a synthetic hydrocarbon molecule that consists of eight carbon atoms arranged at the corners of a cube, with one hydrogen atom attached to each carbon atom. A solid crystalline substance, cubane was first synthesized in 1964 by Philip Eaton and Thomas Cole. Prior to this work, researchers believed that cubic carbon-based molecules would be too unstable to exist.

Taken from bitrebels.com

Taken from bitrebels.com

On the fun side, the cube was morphed—so to speak—into what became an amazingly popular game, or should I say, object of competition. Rubik’s Cube is a 3-D combination puzzle invented in 1974 by Hungarian sculptor and professor of architecture Ernő Rubik. If you’re wondering about the world’s record for solving this puzzle, it’s currently a mind-boggling 4.9 sec, according to a list (with video links) of this and past records that appear to have been broken regularly, just as I opined at the beginning of this blog. But I digress…yet again.

Nucleic Acid-Based Circulating Biomarkers for Cancer Diagnostics Become Reality

  • Circulating Tumor Cell Blood Tests Approved by FDA
  • Circulating DNA Stool Test Approved for Colorectal Screening to Avoid Colonoscopy
  • Circulating mRNA Urine Test Approved for use to Reduce the Total Number of Unnecessary Prostate Biopsies

Backstory

Taken from sysmex-inostics.com 

Taken from sysmex-inostics.com

According to the NIH National Cancer Institute website, ~1.6 million persons in the U.S. alone will be diagnosed with cancer this year. A very important key to survival is early detection. To enable significantly earlier diagnosis compared to manifestation of clinical symptoms, researchers have been focusing on finding DNA or RNA biomarkers that are circulating in blood, which is readily available and relatively noninvasive compared to traditional biopsies.

exosomesSome of the basic processes underlying this paradigm-shift in cancer diagnostics are depicted in the simplified cartoon wherein tumor cells, or components thereof, pass into the bloodstream. This leads to circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) to investigate and differentiate from their normal counterparts as sources of potential biomarkers.

That task is much easier said than done because of the need to sort through all of the normal components in blood, as well as deal with circulating cells and DNA derived from apoptosis (aka programed cell death) and necrosis that are normal ongoing “background” to contend with. In addition to CTCs and ctDNA, there is active cellular excretion of small (30-100 nm) exosome particles as depicted in the following graphic. Consequently, gene-encoding mRNAs, gene-regulating micro RNAs (miRNA), and potentially other exosomal components, can serve as diagnostic biomarkers.

Snapshots of Recent Commercial Diagnostic Products

My search of PubMed for publications indexed to “circulating biomarkers” AND “cancer” led to ~9,000 items, the vast majority of which have appeared during the past decade at an accelerating annual rate.  In fact, there were ~1,000 publications in 2014 alone—that’s roughly 3 such publications every day! Those interested in perusing this mountain of information later can use this link, as my intention here is to comment on resultant commercial diagnostic products, each of which provides all-important early diagnosis using a simple blood test, or urine or stool.

CTCs

In one of my blogs last year, I asserted that liquid biopsies were (metaphorically) clinically valuable “liquid gold” in a modern day Gold Rush. My evidence for the “rush” was a then recent review in Clinical Chemistry stating that “the detection and molecular characterization of CTCs are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker.” In that vein—double pun intended—who’s struck it rich, so to speak, commercially?

Taken from journal.frontiresin.org

Taken from journal.frontiresin.org

The answer is Veridex, which developed the CELLSEARCH® CTC Test that has the added distinction of being the first FDA-approved in vitro diagnostic (IVD) test for capturing and counting CTCs to determine the prognosis of patients (in this case for metastatic breast, colorectal or prostate cancer). This test utilizes magnetic capture of cancer-specific antibodies as depicted below.  Veridex was subsequently acquired by Jansen Diagnostics, which now offers a complete system for CELLSEARCH® CTC Test comprising sample collection, sample preparation, and sample analysis using unique immuno-magnetic and fluorescence imaging technology.

In addition, a Swiss molecular diagnostics company, Novigenix, offers its blood tests for early detection of cancer. Colox®, its lead product, is designed to significantly reduce mortality from colorectal cancer through early detection and follow-up colonoscopy. Novigenix’s technology is based on predictive gene expression profiles of circulating blood cells and tumor-derived protein markers.

Taken from Soper and coworkers in Chem. Commun. (2015).

Taken from Soper and coworkers in Chem. Commun. (2015).

Although not yet a diagnostic device, Prof. Steven Soper at UNC-Chapel Hill and a team of coworkers have recently published methods whereby captured CTCs can be enzymatically released for further analysis. This release procedure (depicted right) features use of an oligonucleotide linker containing uracil (U) that is cleaved by USER™, which consists of a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.

ctDNA Biomarkers for Colon Cancer Screening

That ctDNA can provide promising biomarkers for noninvasive assessment of cancer has been successfully translated into a commercial product by Trovagene, which tests for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagnostic value of ctDNA.

In addition, Cologuard® (developed by Exact Sciences in Madison, WI) was approved by the FDA as the first stool-based colorectal screening test that detects red blood cells and DNA mutations that may indicate colon cancer or precursors to cancer. Its commercials are frequently seen on TV. Given the inconvenient colon-cleansing required of patients prior to the also unpleasant invasiveness of colonoscopy, it’s not surprising that more and more persons are opting to use this new test.

In fact, Exact Sciences recently reported that during the first quarter of 2016, the company completed approximately 40,000 Cologuard® tests, an increase of more than 260% compared to approximately 11,000 tests completed in the same quarter of 2015. The cumulative number of physicians ordering Cologuard® since launch expanded to more than 32,000. Finding a doctor is relatively easy, as I found out when I located a gastrointestinal (GI) specialist near me who was also in my network—yeh!

Given the high incidence rate of colon cancer, and the traditionally recommended screening process, it was necessary for Exact Sciences to obtain compelling data in a large clinical study. An FDA announcement stated that the safety and effectiveness of Cologuard® was established in a clinical trial that screened 10,023 subjects. The trial compared the performance of Cologuard® to the fecal immunochemical test (FIT), a commonly used non-invasive screening test that detects blood in the stool. Cologuard® accurately detected cancers and advanced adenomas more often than the FIT test.

Other ctDNA Biomarkers

PlasmaSelect-R™ offered by Personal Genomics Diagnostics, which is a service company founded by experts at Johns Hopkins University, analyzes ctDNA in blood for genetic alterations in cancer based on a targeted panel of 63 well-characterized cancer genes. Cell-free DNA is extracted from plasma using proprietary methods for low-abundance sample DNA, and processed using a proprietary capture process for high-coverage next-generation sequencing to allow tumor specific mutations, amplifications, and translocations to be identified with a high sensitivity (allele fractions as low as 0.10%) and specificity. The company states that its “services further the understanding of cancer and facilitate the development of new diagnostics and therapeutics through our pioneering research approaches and novel technologies.” 

In June 2016, Roche announced that the FDA approved the cobas® EGFR Mutation Test v2 for use with plasma samples, as a companion diagnostic for the non-small cell lung cancer (NSCLC) therapy, Tarceva®. It’s important to recognize that this is the first FDA approval of a liquid biopsy test as an aid in clinical decisions, and makes it the only companion diagnostic that is FDA-approved for the detection of the epidermal growth factor receptor (EGFR) gene in tumor DNA derived from plasma (or tumor tissue). NSCLC patients who have EGFR exon 19 deletions or L858R mutations are candidates for the EGFR-targeted therapy Tarceva® (erlotinib) in first-line treatment.

Circulating RNA and miRNA

The discoveries in 1999-2000 of tumor-derived RNA in the blood of cancer patients sparked a new field for studying gene expression noninvasively using quantitative reverse transcription-PCR (qRT-PCR) and then next-generation sequencing. The existence of circulating RNA was surprising because ribonucleases are present in blood. However, mechanisms that protect circulating RNA reportedly include complexation to lipids, proteins, lipoproteins, or nucleosomes, and protection within apoptotic bodies or other vesicular structures.

Cleverly named Molecular Stethoscope is a newish startup co-founded by uber-famous Drs. Stephen Quake and Eric Topol. The company has leveraged Quake’s finding that genome-wide analysis of circulating RNA shows tissue-specific signatures from all of the major organs can be monitored in blood, and Topol’s finding that such signatures can be used to predict imminent occurrence of a heart attack. Coronary artery disease, neurodegenerative diseases, and autoimmune/inflammatory diseases are the company’s current objectives. I’m guessing, however, that cancer might be added or licensed.

My search of the literature indicates that there are far more publications on circulating miRNA, presumably due to its greater abundance resulting from its small size and/or binding to miRNA-related proteins. The biogenesis of miRNA is depicted below.

Taken from nature.com

Taken from nature.com

A review and prospectus for circulating miRNA applied to cancer has been recently published by Bertoli et al. in an article entitled MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer. From my search of this emerging field, some exemplary commercial endeavors are as follows.

The first blood-based cancer diagnostic to exploit exosomes became commercially available in the U.S. in January 2016 via launch of ExoDx Prostate(IntelliScore) by Cambridge, MA-based Exosome Diagnostics. As reported by a large team of medical experts in JAMA Oncology, qRT-PCR was used to compare the urine exosome 3-gene expression with biopsy outcomes in patients with a range of low-to-high prostate-specific antigen (PSA) levels (2 to20 ng/mL).

Taken from nature.com

Taken from nature.com

The investigators concluded that this qRT-PCR assay using urine was associated with improved identification of patients with higher-grade prostate cancer among men with elevated PSA levels and could reduce the total number of unnecessary biopsies from the ~1M total annual biopsies. The complications that have been associated with unnecessary biopsy and overtreatment range from erectile dysfunction and incontinence, to infections, sepsis and serious cardiovascular events.

At the other end of the commercial spectrum, so to speak, startup Miroculus aims to aid in the early diagnosis of cancer by making a low-cost, open-source, decentralized diagnostic they called Miriam pictured below. Their goal is for untrained workers in clinics around the world to be able to use Miriam to screen for cancer.

Taken from miroculus.com

Taken from miroculus.com

Miriam made its—or more gender specific—her public debut at the TEDGlobal conference in Rio De Janeiro in 2014 with TED curator Chris Anderson calling it ‘one of the most thrilling demos in TED history’, according to Miroculus. To see and hear why this opinion is accurate, and how Miriam will work in concert with a smartphone camera and cloud interface, I urge you to check out the ~11 minute TEDGlobal presentation at this link, which also gives a short, layperson introduction to miRNA biomarkers in blood for cancer.

Oh, One More Thing

Taken from graymatters.com

Taken from graymatters.com

Although this post focuses on nucleic acids, it’s worth noting that protein biomarkers in blood are also being investigated. In view of increased awareness and media attention about concussion injuries in the National Football League (NFL), a timely example of protein biomarkers for diagnosis of chronic traumatic encephalopathy (CTE)—which heretofore has not been possible by any test—is in development.

Currently the only way to diagnose CTE is through a post-mortem autopsy, but Aethlon Medical Inc. intends to change that with the diagnostic test being developed by its subsidiary Exosome Sciences. The test being studied is designed to identify an abnormal protein called tau that builds up in brain tissue as a result of repetitive head trauma. CTE researchers believe that they have developed a means of measuring plasma exosomal tau. Researchers thought that exosomes had potential as a means of identifying CTE because they cross the blood-brain barrier and can provide a unique method of measuring certain aspects of the contents of brain cells through a blood test.

Exosome Science was able to use its diagnostic blood test in 78 NFL players with histories of concussions, as well as in a control group made up of 16 athletes involved in non-contact sports. The subjects are all part of a much larger NIH-funded project called DETECT, which is focused on developing a variety of biomarkers for CTE and involves researchers at Boston University School of Medicine and the University of Washington.

Look for a future post here about DETECT involving nucleic acid biomarkers.

As always, your comments are welcomed.

Genes in Space

  • High School Student’s PCR Experiments Launched to Space Station
  • Program Evaluates Epigenetics Linked to Astronaut’s Altered Immunity
  • Amplyus Has Big Plans for Its Tiny, Low-Cost PCR Device

In my 2013 blog post on the 30th anniversary of the invention of Nobel Prize-winning PCR by Kary Mullis, I ventured to say that PCR of DNA or RNA was the most widely used—and enabling—method for all life sciences on planet Earth. This accolade can now be expanded to extraterrestrial space in view of PCR experiments to be carried out in the International Space Station (ISS) following the April 8th launch from Kennedy Space Center in Florida aboard NASA’s Cargo Resupply Services flight (CRS-8).

Taken from earthkam.org

Taken from earthkam.org

What makes this “out of this world” milestone for PCR even more exciting is that it’s the result of competition among students in high school—yes, high school—to conceive and design PCR-based studies relevant to living in space. Following is a brief synopsis of the program, the winning high school student, and a small startup company with big plans for its low-cost miniPCR™ device.
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World’s Smallest Real-Time PCR Device

  • Fits in the Palm of your Hand, and Has Single-Molecule Sensitivity
  • Analyzes 4 Samples, and Can be Modified to do 8 
  • Project Leader Reveals Commercialization Details 

I think we’re all fascinated by catchy headlines touting the world’s biggest, tallest, etc., so a recent publication by Ahrberg et al. in venerable Lab on a Chip claiming the world’s smallest real-time PCR device instantly struck me as blogworthy. It also seemed quite apropos as a follow-up to my previous blogs on the continuing shrinkage, so to speak, of real-time PCR technology for point-of-care qPCR diagnostics or other emerging applications in the field.

This hand-held real-time PCR device, developed by A*STAR Singapore, is amazingly small in comparison to the first real-time PCR system introduced by Applied Biosystems in 1995 that weighed 350 pounds and had a width of 7 feet, thus requiring an entire bench top.

pcr

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

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Norovirus: Science Behind the Headline

  • The Virus is Quite Common with 267 Million Cases and 200,000 Deaths Annually
  • RT-PCR is the Detection Method of Choice
  • First Cell-Culture System May Speed Drug and Vaccine Development

We’ve all seen TV news stories about disgruntled passengers disembarking cruise ships returning to port early because of an outbreak of nasty gastroenteritis (i.e. inflammation of the stomach and intestines leading to nausea, vomiting, diarrhea, and stomach cramps). Norovirus (NoV) is the causative agent of these frequently reoccurring “nightmare” cruises, of which 13 have been reported since 2012, sickening some 200-600 passengers. It’s not just limited to cruises, the virus affected 100+ students at a school in Eugene, Oregon last year. And now there’s new evidence for transmission of NoV by eating oysters—which I will therefore not eat in the future.

Taken from counselheal.com.

Taken from counselheal.com.

But perhaps the most NoV-related media attention—and investor ire or litigant action—has been recently focused on gastroenteritis outbreaks at Chipotle—a popular restaurant chain. A criminal investigation is under way at Chipotle, and according to an Associated Press report the company has been served with a federal subpoena.

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‘Genospirituality’—In the Spirit of the Holidays

This last blog for 2015 comes at a time when many of us are looking forward to the upcoming holiday season to enjoy, in various ways, being or sharing with family and friends, and reflecting thankfully for what we have. We often refer to this as getting into “the spirit” of the holidays, regardless of one’s religious or secular beliefs.

In this context, and with the nucleic acids research-relatedness of my blogs in mind, I thought it would be apropos to tell you a bit about some intriguing research aimed at assessing genes associated with spirituality, by which is meant “an inner search for enlightenment achieved through practices such as prayer [religious] or meditation [secular]”, as elaborated elsewhere.

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