Wednesday, January 29, 2014

Genzyme to Present Case Studies on MALDI-TOF for Microbial Identification

Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) mass spectrometry provides an accurate molecular weight measurement and characterization of biomolecules, including proteins, peptides, polysaccharides, nucleic acids and now, microorganisms. Within a few seconds, a mass spectrum can be generated from an isolated colony, providing a microbial identification down to the species level.

In an upcoming free web seminar, Jennifer Reyes will explain how Genzyme, Inc. implemented MALDI-TOF mass spectrometry in a QC microbiology laboratory for the rapid identification of microorganisms.  Organized by SelectScience and Bruker, this complimentary web seminar will provide a starting point for companies wanting to explore alternative and rapid methods for the identification of microorganisms.

Jennifer will present case studies that will cover the following topics:
  • Ability to save time and costs
  • MALDI-TOF performance qualification compared to MicroSEQ and VITEK2
  • Specificity, accuracy and robustness of bacterial extraction methods
  • Method tips for improved extraction of bacterial and fungal samples
Please CLICK HERE for more information and to register.  The free web seminar will take place on February 6, 2014, at 11:00 AM EST.  

Thursday, January 16, 2014

Reviewing Pharmig's 21st Annual Microbiology Conference

After a very successful 2013 annual meeting (see more below), Pharmig is looking to expand it’s scientific programs to include back-to-back microbiology and rapid method conferences in May 2014. The location is currently planned for Ireland and promises to bring the latest in updates and hot topics in pharmaceutical microbiology, regulatory expectations and case studies. Be sure to visit Pharmig’s website ( for updates.

During the 21st annual microbiology conference, which was held in Oxford, UK, more than 100 attendees, speakers and suppliers contributed to lively discussions on what’s happening in the world of pharmaceutical microbiology. The meeting began with an overview of microbiology OOS investigations from a regulatory perspective by Di Morris (ex-MHRA and currently a Consultant Qualified Person). This was followed by Andrew Matthews who expanded on Di’s presentation and provided strategies for effective team investigations.

After an energizing tea and coffee break in the exhibit hall, Barbara Gerten reviewed USP <1117>, microbiology best practices, and supplementation of practical information by ISO standards. She talked about media preparation, storage and shelf life testing, quality control of media, preparation and maintenance of cultures and sample handling. 

Next, attendees had an opportunity to attend one of four open discussion sessions, with topics including microbiological containment through garments, questions you always wanted to ask an inspector, rapid methods and biological indicators.

After lunch, I provided an overview of the myths and misconceptions surrounding the implementation of rapid methods. This included discussions on the impact to industry, regulations and regulatory expectations, validation, Quality by Design, changing acceptance levels and return on investment.

Edel Fitzmaurice and Julie Roberts discussed validation, verification, qualification and suitability of microbiological methods. They reviewed Ph. Eur. and USP definitions, when to validate versus verify, and provided case studies to support their recommendations.

Next, John Hutcheson provided practical considerations when dealing with biofilms in water systems. He discussed the risks to water quality, and how to control, remove and measure biofilm in these systems.

Day one closed and the gala dinner and dance began. If you have never been to a Pharmig meeting before, the social event provides for one of the best networking opportunities in the industry! And during the meeting, Dr. Tim Sandle was awarded Pharmig’s highest honors.

Day two began with Stephen McGrath’s review of objectionable organisms, compendial positions, regulatory perspectives and new emerging organisms that have caused issues within the industry.

Tim Sandle then presented examples of fungal contamination in pharma products, contamination recalls, cleanroom and operator sources, environmental monitoring and disinfectant testing.

Next, Paul Newby provided an update on the latest draft of USP <1115>, bioburden control of non sterile drug substances and products. He also shared his views on what should be considered during product development in addition to nonsterile manufacturing. 

Following lunch, Simon Richards discussed microbiological issues associated with medical devices, including cleaning validation, risk management and the significance of product bioburden.

The final presentation was from Anthony Hilton, Head of Biology & Biomedical Science, Aston University. Dr. Hilton challenged the “5 second rule,” you know, when you drop food on the floor and as long as you pick it up within 5 seconds it is safe to eat? Well, for the first time, he presented scientific data on the microbiological aspects of what happens when food lands on the floor and carpet, and based on the food itself, we learned what we should be putting in our mouths and what we should avoid. A very stimulating discussion to end the meeting (and right before tea and treats)!

If you missed this meeting I would encourage you to attend future Pharmig conferences. I found the caliber of speakers, the relevant topics and networking opportunities to be among the best being offered today.

Sunday, January 12, 2014

USP Rapid Method Update Disappoints at ECA Conference

During the recent ECA Rapid Microbiological Methods conference, Dr. Tony Cundell (Vice Chair of the USP Microbiology Committee of Experts) provided an update on the revision to USP informational chapter <1223> and the development of a new compendial rapid sterility test chapter. The presentation left attendees with more questions than answers, and for some rapid method suppliers and end-users, a disappointment in the direction the USP is taking.

Dr. Cundell first described the objective of a proposed revision to chapter <1223>, Validation of Alternative Microbiological Methods.  The revised chapter is intended to provide general guidance in the implementation of alternative methods for compendial microbial tests. He stated that the chapter would address a number of factors that must be considered prior to the selection of equipment and the qualification of assays with actual product. The factors to consider include the identification of suitable alternate assay methodologies, the development of user-specified stations for the selected equipment, demonstration of the applicability of the method as a replacement for a standard compendial method, installation and operational qualification of equipment, and laboratory qualification of the test method.

Next, Dr. Cundell provided an outline of the individual chapter sections. These include user requirements, components of data quality, instrument qualification, installation, operational, and performance qualifications, demonstration of equivalency, traditional validation criteria for quantitative and qualitative tests, user and instruments supplier responsibilities, method suitability, equivalency, statistical tools, and key references. 

It is of interest to note, that a number of these proposed chapter sections have already been addressed in the recently published revision to PDA Technical Report No. 33 (click here for an overview). However, when Dr. Cundell was asked if the teachings from TR33 would be considered, his response was that there might be diverging guidance from the technical report (and even Ph. Eur. 5.1.6, which is also in revision) because as an informational chapter, the USP does not have to harmonize with other documents. This statement was a surprise to many of the conference attendees, given the fact that the guidance in TR33 represents industry best practices for the validation and implementation of rapid methods, and that the strategies utilized by multinational firms for the regulatory approval of such technologies have been incorporated into the new TR33.

When discussing method equivalency, Dr. Cundell referenced a stimuli article that was previously published in the Pharmacopeial Forum (Hauck et al., 2009, Acceptable, equivalent or better approaches for alternatives to official compendial procedures. Pharm. Forum. 35(3): 772-778). He presented Hauck’s “comparison to alternative method options” matrix that would be included in the revised chapter <1223> for the purpose of providing guidance on determining whether an alternative method was equivalent or better to the compendial method. Unfortunately, Dr. Cundell did not spend sufficient time on this topic for the attendees (and this author) to fully understand the impact of this matrix on the validation of alternative or rapid microbiological methods. We are hopeful that this will be thoroughly explained when the revised chapter <1223> is available for review.

Next, Dr. Cundell provided a brief history of the plate count technique and the colony forming unit (CFU), including limitations when using this microbial viability “signal.” This included the inability of microorganisms to grow in a defined medium. For example, species that would normally be culturable may fail to grow because their growth state in nature, such as dormancy, prevents adjustment to conditions found in the medium.  He then described microbial viability signals other than the CFU, including ATP levels, PCR amplified target sequences, autofluorescence via flow cytometry, and the detection of cells stained with viability markers. In any case, Dr. Cundell stated that to take advantage of emerging technologies, we might need to cut the ties to the CFU as a so-called gold standard. Interestingly, there are a number of alternative and rapid technologies currently available that still rely on the growth of microorganisms, especially when large numbers of organisms are required to generate an alternate viability signal. An example may be found with most ATP bioluminescence systems, in that a single bacterial cell does not produce a sufficient quantity of ATP to be directly detected by these technologies.

When asked about whether specific recommendations were to be provided with regard to validation criteria, Dr. Cundell noted that internal USP statisticians were working on appropriate models and that these would be communicated in a draft in-process revision that will be available for review and public comment sometime in mid-2014.  We are hopeful that the USP takes current industry best practices into consideration, such as those that are provided by PDA TR33 (which, by the way, have been accepted by worldwide regulators) and not to completely reinvent the wheel. We will just have to wait and see.

The second half of Dr. Cundell’s presentation concentrated on the development of a new rapid sterility test chapter; however, the focus was on a rapid sterility test for short-lived, cell-based biological and radioactive injectable products. For these products, the USP has designated that a rapid sterility test be completed in 48 hours, preferably in real time, is non-proprietary, can be conducted in any competent microbiology laboratory, and would meet the method validation requirements as outlined in chapter <1223>.  Technologies that the USP committee considered include ATP bioluminescence, carbon dioxide detection, advanced imaging, flow or solid phase cytometry, and PCR.

Surprisingly, the USP rejected ATP bioluminescence and carbon dioxide detection as not being suitable because they are growth-based with an “incubation time of the order of 7 days.” However, Dr. Cundell stated that these could still be validated as alternative methods using the validation criteria provided in USP <1223>.  It is of interest to note that a number of cell-based biologic manufacturers have already obtained regulatory approvals for using carbon dioxide detection technologies as alternatives to the compendial sterility test, and ATP bioluminescence has also been validated as an alternative sterility test for conventional pharmaceuticals with a less than 7-day incubation period. And the USP ignored solid phase cytometry, which has been validated and approved (FDA and EMA) as an alternative sterility test within our industry, with single cell detection and a time to result in about 4-6 hours.

Moving forward, Dr. Cundell stated that the USP chose two technologies that will be developed for a rapid sterility test: flow cytometry of vital stained microbial cells and RT-PCR using universal primers and probes.

I question this decision.  And so did many rapid method suppliers who attended Dr. Cundell’s presentation.

For instance, knowing that flow cytometry will not necessarily provide single cell detection, and that this method can only analyze small volumes during the test (e.g., 1 mL or less), there will need to be an appropriate enrichment or growth period (of a suitable amount of the test sample) to allow for single cells to proliferate to a level that can be detected by flow cytometry. It is unclear whether this can occur within the USP’s 48-hour rapid sterility test requirement.  Incidentally, ATP bioluminescence may also be used under this same scenario (enrichment followed by detection), but ATP systems were rejected by the USP as being inappropriate for a rapid sterility test methodology.  

The same may be said for RT-PCR.  If the starting material for this test is DNA, then it will be necessary to enrich the sample to generate a sufficient number of target sequences to be amplified by the PCR system. Otherwise, the potential for false positives due to the presence of residual DNA (from dead cells and/or cross contamination) may increase.  And it is also unclear how long the enrichment period will be required for in order to detect all types of microorganisms, including those are fastidious, slow growing or injured.  

Dr. Cundell concluded his presentation by stating that the next step will be the development of a draft chapter, Rapid Sterility Tests, numbered 71.1. [Author note: because the chapter number will be less than 1000, this would be a compendial procedure for which compliance is enforceable by the FDA.]

Dr. Cundell also stated that protocols would be developed for external collaborators to conduct proof of concept studies to demonstrate the robustness of the proposed methods. And based on these experiences, the chapter will be finalized and published as an in-process revision for public review and comment.

Saturday, January 11, 2014

ECA Rapid Methods Conference a Huge Success

In December 2013, the European Compliance Academy (ECA) hosted their annual rapid microbiological methods conference and it was, as is usually the case, a huge success! Axel Schroeder and Jessica Sturmer, both of Concept Heidelberg, along with Program Chairperson Dr. Sven Deutschmann, welcomed attendees to Barcelona, Spain, for this yearly event.

The two-day conference and an additional workshop on statistics provided attendees with a unique opportunity to evaluate the new developments in RMM systems, experiences in validation as well as implementation within the pharmaceutical industry. Attendees also learned about the expectations of authorities and recent developments in regulatory requirements. Experts, end-users and suppliers provided insights in the routine use of RMMs and during the statistics workshop, practical examples and information about the interpretation of microbiological validation data.

The conference opened with Dr. Tony Cundell providing an overview of the USP’s revision to chapter 1223 and the development of a new rapid sterility test chapter.  This opening presentation was very provocative and provided for quite an interesting Q&A session, especially from RMM technology suppliers and validation experts.  Some were left scratching their heads trying to understand the direction the USP is taking, and this is the topic of a more comprehensive discussion I provided in a separate blog posting (click here to read).

Stephen Wicks (EDQM) then provided an overview of the current status of the revision to European Pharmacopoeia chapter 5.1.6.  Based on his discussions, the draft chapter appears to be better aligned with the new PDA Technical Report No. 33. Additionally, new validation examples will be proposed which will include rapid sterility testing using membrane filtration, a quantitative assay for the enumeration of microorganisms using solid phase cytometry, and a PCR-based microbial identification method. It is anticipated that the working party will meet in early 2014 to finalize the revision, followed by a public inquiry in Pharmeuropa. Following public comment, the working party will put the finishing touches on the draft, with a mid to late 2014 publication date.

Following Stephen’s presentation I presented an overview of the revision to PDA Technical report No. 33, which was published in October 2013. I recently published this overview in American Pharmaceutical Review, and you can download the article by Clicking Here.

Jeanne Moldenhauer (Excellent Pharma Consulting) followed with a lively discussion on rapid sterility testing and the methods that have been approved by the regulators in early submissions. She also described the reasons why a number of companies became disillusioned with their rapid sterility methods and abandoned their use. She finished with suggestions for how the industry can prevent this type of occurrence from happening in the future.

Next, Judy Madden (Celsis) provided a short presentation on validating a rapid sterility test by direct inoculation using the ATP bioluminescence Celsis Advance System.

This was followed by an end-user’s validation perspective of the Celsis system. Dr. Erik Wilkens (Novartis) discussed a proposed workflow and validation plan for a direct inoculation method in which a non-filterable product or device is enriched in a liquid medium, followed by centrifugation of the media and then introduction into the Celsis system.   

David Jones (Rapid Micro Biosystems) provided a short presentation on the second generation Growth Direct System. He discussed automating bioburden, environmental monitoring and sterility testing using their autofluorescence technology.

This was followed by an end-user’s perspective of the same Growth Direct technology. Oliver Gordon (Novartis) shared their feasibility and neutralization studies, which included processing actual EM samples (active air and surface samples).  The Growth Direct results were comparable with the classical method of visually observing and enumerating CFU’s on agar plates. 

A short presentation by Pall Corporation discussed a case study on factors to consider for rapid method system implementation.

Next, Dr. Marcel Goverde (MGP Consulting) gave an excellent overview of helpful literature and online resources that offer validation and implementation guidance. 

This was followed by another short presentation by a RMM supplier. Tim Russell (TSI) provided an overview of their BioTrak real-time viable particle detection system.  The presentation included a discussion of laser induced intrinsic fluorescence, how to minimize false positive results, and how to utilize the system’s built-in collection filter for subsequent microorganism analysis and identification. 

Dr. Emiliano Toso (Merck Serono) then shared his experiences with in-process control testing by traditional, innovative and next generation methods in order to avoid large-scale virus contamination events, such as those caused by Vesivirus and Mouse Minute Virus.  Focusing on molecular methods, he described reverse transcriptase gene amplification techniques, next generation and deep sequencing methodologies. 

The next speaker was Dr. Franz Gruber (Baxter) who described the long-term experience regarding alternative Mycoplasma testing according to European Pharmacopoeia expectations.  He provided an overview on an RNA-based methodology for nucleic acid extraction and amplification, handling, storage, and stability, and the relationship between the alternative method and reference preparations.

Dr. Sven Deutschmann (Roche Diagnostics) provided a summary of a PCR-based method for the detection of Leptospira.  He described the regulatory expectations (FDA, EMA and the Japanese PMDA) for in-process testing of Leptospira in CHO-cell processes.  Validation approaches for detection limit, specificity, robustness and limiting cross contamination were discussed. 

bioMerieux provided the next vendor short presentation and this focused on their BioBall product.  David Myatt described the very precise flow cytometry and freeze drying manufacturing process that allows the consistent generation of a stable and precise low level inoculum. 

Dr. Edwin van den Heuvel (UMC Groningen) and Dr. Pieta Ijzerman-Boon (MSD) provided an introduction into the statistics workshop that they presented following the main conference.  They discussed statistical detection mechanisms, validation issues and validation experiments. Not for the faint hearted, this intense statistical overview consumed the attendees with guidelines on recommended inoculation levels, replicate numbers, false positives and false negatives, and of course, statistical models.

The final presentation by Dr. Sebastien Ribault provided insights into whether there exists a need for rapid methods as in-process controls during biotech processing.  The impact of using RMMs on projects timelines was also considered.

As you can see from the caliber of expertise of the speakers, this past ECA conference on rapid microbiological methods promoted lively discussions (and definitely debate), the sharing of best practices and most importantly, a sense of camaraderie among fellow scientists and experts in the field of RMMs.

Tropical Medicine Researchers Test New Dengue Detection Kit

University of Hawai`i Mānoa scientists have found that a commercially available, FDA-approved dengue detection kit bests the former “gold standard” test by producing results in under five hours.

A study conducted at the John A. Burns School of Medicine (JABSOM) sought to evaluate the use of the commercially available, Food and Drug Administration (FDA) approved InBios Dengue virus IgM ELISA kit for rapid diagnosis of dengue virus infection. This kit detects anti-dengue virus IgM antibodies, which are produced within three to five days after the onset of dengue fever.

Dengue virus clinical manifestations vary from asymptomatic infection, mild fever to a severe disease characterized by hemorrhage and shock. Dengue virus outbreaks occurred in Hawai`i in 2001 and 2011 with 153 and four dengue cases, respectively.

Rapid diagnosis of dengue virus infection is critical for effective patient management, thus can prevent the severe dengue disease. In addition, it also helps to prevent the spread of dengue virus infection, which can occur after the bite of dengue-virus infected mosquitoes.

In the study, reported in the Journal of Clinical Microbiology, JABSOM’s Department of Tropical Medicine, Medical Microbiology and Pharmacology tested 79 well characterized clinical serum samples collected from Hawai`i, Vietnam, Niue, Singapore and American Samoa, where dengue virus outbreaks occurred in the past, using InBios DENV IgM ELISA kit and results were compared to that of “gold standard’ DENV IgM antibody capture ELISA (MAC-ELISA). The agreement, sensitivity, and specificity of the InBios assay were 94, 92 and 94% respectively. The study found that InBios’ DENV Detect IgM Capture ELISA is advantageous compared to the in-house MAC-ELISA, as the results can be obtained in less than five hours, whereas the in-house MAC-ELISA requires 2 to 3 days. We conclude that InBios DENV IgM Capture ELISA can be effectively used for rapid diagnosis of acute or recent DENV infection.

This study was supported in part by grants P20GM103516, U01AI078213 and U54MD007584 from the National Institutes of Health, grant W81XWH0720073 from the Department of Defense and by institutional funds.

About Dengue

Dengue is a significant human pathogen of global importance. Today about 2.5 billion people, or 40% of the world’s population, live in areas where there is a risk of dengue transmission. Dengue is endemic in at least 100 countries in Asia, the Pacific, the Americas, Africa and the Caribbean. Recent reports indicate that there are about 350 million people infected with the dengue virus annually worldwide, triple the World Health Organization (WHO) estimates of 50 to 100 million annual infections, mostly among children. Recently there have been reported outbreaks in Kenya (May 2013) and Angola (June 2013). Although most of the reported cases in the United States are acquired by travelers or immigrants, autochthonous dengue fever outbreaks have occurred in Brownsville, TX (2005), southern Florida (2009-2011) and Hawai`i (2011).

Source: University of Hawai'i

Sunday, January 5, 2014

A Smartphone Medical Lab

Last year, Max Perelman worried that his 3-year-old daughter's rash could be chicken pox or poison oak. After visiting the pediatrician, he waited for test results, promised within 24 hours, that never came - yet two months later, he received a $35 bill for his share of the care.

As cofounder of a Philadelphia start-up, Perelman is itching to prevent such incidents with Biomeme, a system that pairs with a smartphone to form a disease-diagnostics lab the size of a soda can.

The company wants to democratize rapid, cheap biological testing with quick turnaround for use by health clinics and, perhaps one day, ordinary people from home.

"We see this as a way for people to look at the genetic world, and getting it in the hands of everyday consumers would be really beneficial," cofounder and biologist Jesse vanWestrienen said. "Long term, we want this to be used by everybody in some way."

Biomeme is part of an exploding health trend that aims to exploit the accessibility and built-in capabilities of smartphones. Mobile apps can track our daily sleep cycles, steps walked, calories consumed. They can help locate Alzheimer's patients and assist with the management of diabetes.

Biomeme's system - which includes a sample-prep kit and a sleek phone docking station - wants to take the trend farther, sniffing out the DNA signature of bacteria or viruses in a sample of saliva, blood, or urine. It uses the same proven technology as lab instruments 10 times its size.

Much of its early customer base consists of curious institutions like the U.S. Army and Air Force, which showed interest in using Biomeme for biothreat detection in the field. The Gorgas Institute, Panama's leading public health research center, wants to do tropical-disease biosurveillance with Biomeme's system. And a biochemistry professor at Charles Darwin University in Australia thinks her students could do DNA diagnostic labs at home using Biomeme rather than at university laboratories.

Geneticist Muin Khoury of the federal Centers for Disease Control and Prevention suspects such a device could also be useful for field investigations of outbreaks and large-scale public health studies. But first, Biomeme needs to prove it works on real samples.

"What does a positive test mean? What does a negative test mean? Is it giving accurate results? These are the hard questions that they have to address," he said.

Next year, the company plans to do its first studies with human samples through a collaboration with the Drexel University College of Medicine, using urine from patients with and without gonorrhea. Data from Biomeme will be compared to the diagnostic lab equipment already in use. If all goes well, the physicians will then take Biomeme for a test drive in the women's health clinic to get rapid results for sexually transmitted diseases - a process that usually takes several days.

Biomeme's system consists of two parts: a sample-prep kit and the diagnostics hardware. The sample-prep kit contains test tubes and freeze-dried chemicals color-coded for ease of use that break down cells and purify the genetic material inside.

After a bit of syringing to get rid of unnecessary cellular bits, the search for the needle in a genetic haystack begins. The system uses a technique called quantitative polymerase chain reaction (qPCR) that looks for a specific segment of DNA in the sample - say, that of a flu virus - and, if it is there, makes copies of it using enzymes that react with repeated heating and cooling cycles.

"qPCR is essentially a photocopier for DNA; you can go from one copy of DNA to billions," vanWestrienen said.

By attaching the DNA to a light-emitting molecule, Biomeme can use the phone's camera to monitor the reaction in real time. If the camera detects more light as more copies are made, the sample is positive for flu virus, but if it stays dark, that means flu virus was never present.

 After about 40 cycles, there would be enough DNA snippets to make them easier to find.

Biomeme uses every part of the smartphone it can, not just the camera. The phone's processor is used to run the raw data through algorithms, and the results are then sent to cloud storage via wireless or cellular connection, along with a GPS-tagged location.

"All of those elements are elements we don't need to include in our hardware," Perelman said. "They are elements that Apple and Samsung are experts at, and why not piggyback on that?"

Because it uses a number of components from the user-provided smartphone, costs are kept low; the device will ultimately cost about $1,000.

In April, the three cofounders of Biomeme - Perelman, vanWestrienen, and engineering guru Marc DeJohn - uprooted their lives in California and Arizona to try their luck as full-time entrepreneurs as part of DreamIt Health Philadelphia, a four-month boot camp that helps start-ups get off the ground. In partnership with Independence Blue Cross and Penn Medicine, it provided Biomeme with $50,000 in initial funding as well as office space and mentorship. Through a mix of crowdfunding and large investors, the company has since amassed more than $1 million.

It has settled in as a resident company of NextFab Studio, a manufacturing hub with everything from electronics labs to 3D printers. There are four full-time employees - the cofounders and a software lead - along with a handful of consultants. The name of the company comes from the obvious biology application ("Bio") plus "meme," a term for any idea that goes viral.

"We want to spread biology to the population just like a meme, to empower consumers and patients," Perelman said. Maybe your child has a sore throat, and you want to check for strep without wading through paperwork and red tape. Or you feel awkward about going to the doctor for a gonorrhea test and would rather do it yourself at home.

However, some medical experts aren't thrilled about the idea of patients one day diagnosing themselves without consulting a physician. The CDC's Khoury, though optimistic about the clinical applications, remains cautious about home-based DNA diagnostics.

"I worry a bit about the home part of it," he said. "I love technology, but I don't embrace it blindly."

"People can't examine themselves," said cardiologist Arthur Feldman, also executive dean at Temple University School of Medicine. "There's importance in receiving the advice from a caregiver - somebody that can talk about risks and benefits, along with whatever treatments are available."

Such concerns will likely keep Biomeme's diagnostics-for-all vision firmly in the realm of pipe dreams, at least for the near future. Historically, the Food and Drug Administration hasn't been too keen on at-home DIY disease testing. For instance, the oral swab-based rapid HIV test OraQuick was approved in 2004 only for trained technician use; it took seven years for an at-home version to hit pharmacy shelves.

"We know that for human health applications, the FDA absolutely has to be involved," vanWestrienen said, who calls the approval process "an arduous task."

They haven't spoken with the FDA, although they have a kind of regulatory Sherpa to help them through the process. But rather than worry about it, Perelman remains excited about overseas prospects for now.

"If we can get cost of goods down very low, it will be affordable very soon for everyone," he said.


Friday, January 3, 2014

Vapor Nanobubbles Rapidly Detect Malaria Through the Skin

Rice University researchers have developed a noninvasive technology that accurately detects low levels of malaria infection through the skin in seconds with a laser scanner. The "vapor nanobubble" technology requires no dyes or diagnostic chemicals, and there is no need to draw blood.

A preclinical study published this week in the Proceedings of the National Academy of Sciences shows that Rice's technology detected even a single malaria-infected cell among a million normal cells with zero false-positive readings.

The new diagnostic technology uses a low-powered laser that creates tiny vapor "nanobubbles" inside malaria-infected cells. The bursting bubbles have a unique acoustic signature that allows for an extremely sensitive diagnosis.

"Ours is the first through-the-skin method that's been shown to rapidly and accurately detect malaria in seconds without the use of blood sampling or reagents," said lead investigator Dmitri Lapotko, a Rice scientist who invented the vapor nanobubble technology. The diagnosis and screening will be supported by a low-cost, battery-powered portable device that can be operated by nonmedical personnel. One device should be able to screen up to 200,000 people per year, with the cost of diagnosis estimated to be below 50 cents, he said.

Malaria, one of the world's deadliest diseases, sickens more than 300 million people and kills more than 600,000 each year, most of them young children. Despite widespread global efforts, malaria parasites have become more resistant to drugs, and efficient epidemiological screening and early diagnosis are largely unavailable in the countries most affected by the disease.

Inexpensive rapid diagnostic tests exist, but they lack sensitivity and reliability. The gold standard for diagnosing malaria is a "blood smear" test, which requires a sample of the patient's blood, a trained laboratory technician, chemical reagents and high-quality microscope. These are often unavailable in low-resource hospitals and clinics in the developing world.

"The vapor nanobubble technology for malaria detection is distinct from all previous diagnostic approaches," said study co-author Dr. David Sullivan, a malaria clinician and researcher at Malaria Research Institute at Johns Hopkins University. "The vapor nanobubble transdermal detection method adds a new dimension to malaria diagnostics, and it has the potential to support rapid, high-throughput and highly sensitive diagnosis and screening by nonmedical personnel under field conditions."

The transdermal diagnostic method takes advantage of the optical properties and nanosize of hemozoin, a nanoparticle produced by a malaria parasite inside red blood cell. Hemozoin crystals are not found in normal red blood cells.

Lapotko, a faculty fellow in biochemistry and cell biology and in physics and astronomy at Rice, and lead co-author Ekaterina Lukianova-Hleb found that hemozoin absorbs the energy from a short laser pulse and creates a transient vapor nanobubble. This short-lived vapor nanobubble emerges around the hemozoin nanoparticle and is detected both acoustically and optically. In the study, the researchers found that acoustic detection of nanobubbles made it possible to detect malaria with extraordinary sensitivity.

"The nanobubbles are generated on demand and only by hemozoin," said Lukianova-Hleb, a research scientist in biochemistry and cell biology at Rice. "For this reason, we found that our tests never returned a false-positive result, one in which malaria was mistakenly detected in a normal uninfected cell."

  • The picture above shows how a laser pulse creates a vapor nanobubble in a malaria-infected cell and is used to noninvasively diagnose malaria rapidly and with high sensitivity. Click the picture for a larger image. Credit: E. Lukianova-Hleb/Rice University.

Abstract of Proceedings of the National Academy of Sciences Paper: 

Successful diagnosis, screening, and elimination of malaria critically depend on rapid and sensitive detection of this dangerous infection, preferably transdermally and without sophisticated reagents or blood drawing. Such diagnostic methods are not currently available. Here we show that the high optical absorbance and nanosize of endogenous heme nanoparticles called “hemozoin,” a unique component of all blood-stage malaria parasites, generates a transient vapor nanobubble around hemozoin in response to a short and safe near-infrared picosecond laser pulse. The acoustic signals of these malaria-specific nanobubbles provided transdermal noninvasive and rapid detection of a malaria infection as low as 0.00034% in animals without using any reagents or drawing blood. These on-demand transient events have no analogs among current malaria markers and probes, can detect and screen malaria in seconds, and can be realized as a compact, easy-to-use, inexpensive, and safe field technology.


Ekaterina Y. Lukianova-Hleb et al., Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria, Proceedings of the National Academy of Sciences, 2013, DOI: 10.1073/pnas.1316253111