FDA, CDC, and CMS Launch Task Force to Facilitate Rapid Availability of Diagnostic Tests During Public Health Emergencies

The U.S. Food and Drug Administration, Centers for Disease Control and Prevention (CDC) and the Centers for Medicare and Medicaid Services (CMS) announced the launch of the Tri-Agency Task Force for Emergency Diagnostics. This task force has been created to help leverage the expertise of each agency to advance rapid development and deployment of diagnostic tests in clinical and public health laboratories during public health emergencies.

“Public health emergencies, like Ebola outbreaks, remind us that we’re a global community when it comes to public health protection. Bacteria and viruses don’t respect territorial boundaries. It takes a sustained, robust and globally coordinated effort to protect our nation and the global community from various infectious disease threats. We’re all in this together. To that end, the FDA knows that collaborating with our federal partners to employ our collective expertise, experiences from previous incidents, and resources will better assist in a global response. We also believe that this task force could lead to more innovation for diagnostic tests as developers will see a more predictable federal regulatory response through the agencies’ coordination,” said Jeffrey Shuren, M.D., J.D., director of the FDA’s Center for Devices and Radiological Health. “This task force will help our agencies better collaborate to prepare for, and respond to, public health threats, including identifying threats and ensuring the appropriate diagnostics are in place to support efforts in the field.”

The FDA, CDC and CMS each play a critical role in responding to public health emergencies, including identifying threats, regulating medical products, and providing oversight for laboratories. The agencies have robust teams of scientists, researchers and policy experts that are dedicated to preparing the U.S. for rapid disaster response.

Diagnostic tests—such as those that can detect pathogens like the Ebola and Zika viruses—can be quickly made available to meet response needs during a crisis through the Emergency Use Authorization (EUA) process. The FDA has authority to issue an EUA for the use of diagnostic tests during public health emergencies, provided criteria are met. The CDC is responsible for providing agent-specific subject matter expertise in epidemiology, laboratory expertise and guidance to clinicians and laboratories responding to the emergency. The CDC and other federal laboratories are often the ones developing new tests to respond to emergency needs. CMS has authority to ensure quality testing at laboratories through the Clinical Laboratory Improvement Amendments (CLIA). CMS provides guidance, even during public health emergencies, to laboratories on meeting CLIA requirements to ensure laboratories produce accurate, reliable and timely results.

Prior to this partnership, feedback from the clinical laboratory community indicated that there was uncertainty about how to implement the diagnostic tests once they received an EUA; particularly, the community was uncertain about meeting CLIA regulations under an EUA to allow labs to start testing specimens.

“During public health emergencies, ensuring the health and safety of patients through quality laboratory testing will remain the focus of CMS,” said Kate Goodrich, director of the Center for Clinical Standards and Quality and CMS Chief Medical Officer. “Timely implementation of EUA diagnostic assays in the US healthcare system is dependent upon laboratories understanding the instructions for use and applying them to the patient samples received for testing. As part of this taskforce, it is our goal to provide clear and consistent guidance to laboratories on the application of CLIA requirements for these emergency assays.”

By standardizing collaboration efforts, the federal partners hope to address issues related to implementation of diagnostic tests authorized for emergency use under an EUA, as well as other unmet needs and gaps in preparing and responding to global health threats. The task force will provide a forum for each agency to coordinate, provide consultation, and improve the availability of diagnostic tests during public health emergencies. In addition, to assist in public health preparedness, the task force will work to define, refine and streamline interagency approaches for the implementation of EUA diagnostic tests. The hope is that the task force will enable an even more efficient federal government response for making diagnostic tests available in the event of a public health emergency.

“Time and time again, we’re reminded that disease knows no borders. While our globalized world and modern transportation help promote economic prosperity, these features also facilitate the spread of emerging infectious diseases,” said Chesley Richards, CDC’s Deputy Director for Public Health Science and Surveillance. “In the past 15 years alone, we’ve faced serious global outbreaks of deadly pathogens. During public health emergencies, it is critical for diagnostic tests to be made available and adopted quickly into clinical and public health laboratories for rapid patient care.”

The FDA, an agency within the U.S. Department of Health and Human Services, protects the public health by assuring the safety, effectiveness, and security of human and veterinary drugs, vaccines and other biological products for human use, and medical devices. The agency also is responsible for the safety and security of our nation’s food supply, cosmetics, dietary supplements, products that give off electronic radiation, and for regulating tobacco products.

“0D” Microarrays Of Live Bacteria Using AC Electrokinetics On A Chip

Bacteria have been around for over a billion years and have developed remarkable capabilities to not only cause deadly infections but also to survive adverse environmental conditions (1, 2). This has led to a rich cellular diaspora with diverse geno- and phenotypic variability that is important to characterize owing to its dire implications on mankind.

Researchers are constantly evolving strategies that allow them to study complex cellular processes like cell-cell interactions, cellular metabolic activity, gene function, response to external stimuli (3-7), host-pathogen interactions, and surface-associated redox activities (8) at a single cell level. The foremost requirements of these single cell test platforms are to distinguish, isolate, manipulate, immobilize, and characterize cells in an easy and reproducible manner.

The conventional methods widely used for single cell isolation include fluorescence- or magnetic-activated cell sorting (FACS/MACS), micromanipulation, microdissection, and manual cell picking. While FACS/MACS need high cellular loads, large sample volumes and specific treatment of cells prior to analysis, the remaining techniques have quite a low throughput and require highly-skilled labor for carrying out the experiments. Other methods for cell capture include ink-jet printing on molecularly-patterned templates (9) or dip-pen nanolithography (10), but they again, suffer from poor reproducibility, high cost, and our inability to reuse the substrate.

With recent advances in the field of microfluidics, manipulation and separation of cells on the basis of their dielectric properties, using external electric fields has garnered significant attention. This approach offers numerous advantages such as a high extent of miniaturization, low sample volume, controlled fluid dynamics, ease of sample handling, high throughput, great precision, automation, and label-free operation.

Motivated by these factors, we exploited an electrokinetic phenomenon called alternating current (AC) dielectrophoresis to collect live bacterial cells into grid-like patterns, with each node containing a single cell (see image below). This electric field-assisted assembly not only provided high spatio-temporal control over several thousands of cells simultaneously, but the dynamically-formed cellular arrays were also fully reversible meaning that both the cells and the chips could be retrieved at the end of each capture cycle. This study was recently published in Biosensors and Bioelectronics (111, 2018, 159–165) and showed that model Gram-negative bacteria S.typhi and E.coli can be trapped inside micropatterned conductive circular well electrodes by applying AC electric fields (5 MHz, 5-20 Vpp) across a few microliters of cell suspension.

Dynamic electropatterning of live bacteria into single microwell arrays using AC electrokinetics. The bacteria were fluorescently tagged to allow visualization. Image courtesy Meenal Goel & Shalini Gupta

Shrinking the size of the individual electrodes down to 5 μm allowed single cells to be captured per well with 90% efficiency. This trapping of cells took place due to positive dielectrophoresis that attracted them toward regions of high electric field intensity (microwells in our case). The cells remained alive during the one-hour-long operation, and the overall collection efficiency (from bulk to the surface) was also found to be around 90%.

The potential biotechnological application of our chip was demonstrated in two ways. First, the two cell types were mixed in suspension and collected together using the chip. Counting their relative concentration in the 2D matrix using their phenotypic traits allowed us to directly estimate cellular bulk concentrations in the mixture without generating any calibration curves. Both the total time taken and the volume of the sample used was a fraction of the conventional methods. In the second case, the chip was integrated with an impedance spectroscope to carry out rapid cell viability testing. The response of dielectrophoretically-trapped bacteria against a known antimicrobial peptide was recorded at different drug concentrations and found to be ultrasensitive, reducing the overall time for drug susceptibility testing to just under an hour. This is a significant reduction over standard clinical methods that take several hours to produce outcomes.

In the future, our technology could be used for a wide range of applications including multiplexed sensing, rapid single bacterial profiling in heterogeneous populations, and ultrafast drug susceptibility testing to reduce the use of broad-spectrum antibiotics and, hence, the emergence of new antibiotic-resistant strains.

These findings are described in the article entitled Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing, recently published in the journal Biosensors and Bioelectronics. This work was conducted by Meenal Goel, Abhishek Verma, and Shalini Gupta from the Indian Institute of Technology Delhi.

Source: Science Trends

References:

1. Turner, N. A., Harris, J., Russell, A. D., Lloyd. D. 2000. J. Appl. Microbiol. 89, 751–759.
2. Davey, M. E., O’Toole, G. A., 2000. Microbiol. Mol. Biol. Rev. 64, 847–867.
3. Flaim, C.J., Chien, S., Bhatia, S.N., 2005. Nat. Methods 2, 119–125.
4. Hung, P.J., Lee, P.J., Sabounchi, P., Lin, R., Lee, L.P., 2005. Biotechnol. Bioeng. 89, 1–8.
5. Lee, M.Y., Dordick, J.S., 2006. Curr. Opin. Biotechnol. 17, 619-27.
6. van der Meer, J.R., Belkin, S. 2010. Nat. Rev. Microbiol. 8, 511–522.
7. Ziauddin, J., Sabatini, D.M., 2001. Nature 411, 107–10.
8. Potma, E. O., de Boeij, W. P., van Haastert, P. J. M., Wiersma., D. A. 2001. Proc. Natl. Acad. Sci. 98, 1577–1582.
9. Xu, Luping, Robert, Lydia, Ouyang, Qi, Taddei, François, Chen, Yong, Lindner, Ariel B., Baigl, Damien, 2007. Nano Lett. 7, 2068–2072.
10. Kim, J., Shin, Y.-H., Yun, S.-H., Choi, D.-S., Nam, J.-H., Kim, S.R., Moon, S.-K., Chung, B.H., Lee, J.-H., Kim, J.-H., Kim, K.-Y., Kim, K.-M., Lim, J.-H., 2012. J. Am. Chem. Soc. 134, 16500–16503.

Glow-in-the-Dark Paper as a Rapid Test for Infectious Diseases

Researchers from Eindhoven University of Technology (The Netherlands) and Keio University (Japan) present a practicable and reliable way to test for infectious diseases: All you need are a special glowing paper strip, a drop of blood and a digital camera, as they write in the scientific journal Angewandte Chemie. Not only does this make the technology very cheap and fast -- after twenty minutes it is clear whether there is an infection -- it also makes expensive and time-consuming laboratory measurements in the hospital unnecessary. In addition, the test has a lot of potential in developing countries for the easy testing of tropical diseases.

The image above shows research leader Maarten Merkx with one copy of the 'glow-in-the-dark' test. Photo: Bart van Overbeeke.

The test shows the presence of infectious diseases by searching for certain antibodies in the blood that your body makes in response to, for example, viruses and bacteria. The development of handy tests for the detection of antibodies is in the spotlight as a practicable and quick alternative to expensive, time-consuming laboratory measurements in hospitals. Doctors are also increasingly using antibodies as medicines, for example in the case of cancer or rheumatism. So this simple test is also suitable for regularly monitoring the dose of such medicines to be able to take corrective measures in good time.

Paper gives light

The use of the paper strip developed by the Dutch and Japanese researchers is a piece of cake. Apply a drop of blood to the appropriate place on the paper, wait twenty minutes and turn it over. "A biochemical reaction causes the underside of paper to emit blue-green light," says Eindhoven University of Technology professor and research leader Maarten Merkx. "The bluer the color, the higher the concentration of antibodies." A digital camera, for example from a mobile phone, is sufficient to determine the exact color and thus the result.

This paper strip (extremely zoomed in) contains two copies of the test. The three glowing dots per test indicate that you can check on three different antibodies within one test. Credit: Bart van Overbeeke.

Sensor protein

The color is created thanks to the secret ingredient of the paper strip: a so-called luminous sensor protein developed at TU/e. After a droplet of blood comes onto the paper, this protein triggers a reaction in which blue light is produced (known as bioluminescence). An enzyme that also illuminates fireflies and certain fish, for example, plays a role in this. In a second step, the blue light is converted into green light. But here comes the clue: if an antibody binds to the sensor protein, it blocks the second step. A lot of green means few antibodies and, vice versa, less green means more antibodies.

Market launched within a few years

The ratio of blue and green light can be used to derive the concentration of antibodies. "So not only do you know whether the antibody is in the blood, but also how much," says Merkx. By measuring the ratio precisely, they suffer less from problems that other biosensors often have, such as the signal becoming weaker over time. In their prototype, they successfully tested three antibodies simultaneously, for HIV, flu and dengue fever. Merkx expects the test to be commercially available within a few years.

Now the production of the paper strips is still handicraft. Photo: Bart van Overbeeke.

Journal Reference:

Keisuke Tenda, Benice van Gerven, Remco Arts, Yuki Hiruta, Maarten Merkx, Daniel Citterio. Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins. Angewandte Chemie International Edition, 2018

Abstract:

This work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.

Source: Eindhoven University of Technology

FDA Promotes Rapid Sterility Testing for Human Gene Therapy Products in its Draft Guidance for Investigational New Drug Applications (INDs)

This week, FDA’s Center for Biologics Evaluation and Research (CBER) published six draft guidances relating to gene therapy, three of which cover products for specific disease categories (hemophilia, rare diseases, and retinal disorders), and three of which address manufacturing and clinical study design issues related to gene therapy: chemistry, manufacturing and control (CMC) information in INDs, long term follow-up study design, and testing of retroviral vector-based products.

In a press release, FDA Commissioner Scott Gottlieb, M.D., highlighted “rapid advancements” and “great promise” in the gene therapy space, saying the guidances “are aimed at fostering developments in this innovative field.”  Dr. Gottlieb acknowledged that for some gene therapies, FDA “may need to accept some level of uncertainty” at the time of approval regarding questions related to durability of response, as well as product manufacturing and quality.  He acknowledged the need, however, to assure patient safety and to assure that potential risks are adequately characterized and benefits are adequately demonstrated.

The draft guidance is titled, "Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). Draft Guidance for Industry. July 2018. Clicking on the link will bring up a complete PDF of the guidance document.

The purpose of the draft guidance is to inform sponsors how to provide sufficient CMC information required to assure product safety, identity, quality, purity, and strength (including potency) of the human gene therapy investigational product.

When finalized, the draft guidance will supersede the document entitled “Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),” dated April 2008.

Because the draft guidance recommends the use of rapid microbiological methods (RMM) for in-process and finished product sterility testing, it is important to understand FDA's position in this regard. As such, I will highlight specific sections in the draft guidance and where appropriate, provide additional interpretation and comment.

Introduction

Human gene therapy products are defined as all products that mediate their effects by transcription or translation of transferred genetic material or by specifically altering host (human)genetic sequences. Some examples of gene therapy products include nucleic acids, genetically modified microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, and ex vivo genetically modified human cells. Gene therapy products meet the definition of “biological product” in section 351(i) of the Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the prevention, treatment, or cure of a disease or condition of human beings.

The FDA requires all sponsors of investigational new drug products (DPs), including investigational gene therapy products, to describe the CMC information for the drug substance (DS). FDA may place the IND on clinical hold if the IND does not contain sufficient CMC information to assess the risks to subjects in the proposed studies. The CMC information submitted in an IND is a commitment to perform manufacturing and testing of the investigational product, as stated. However, FDA acknowledges that manufacturing changes may be necessary as product development proceeds, and sponsors should submit information amendments to supplement the initial information submitted for the CMC processes. The CMC information submitted in the original IND for a Phase 1 study may be limited, and therefore, the effect of manufacturing changes, even minor changes, on product safety and quality may not be known. Thus, if a manufacturing change could affect product safety, identity, quality, purity, potency, or stability, sponsors should submit the manufacturing change prior to implementation.

What is Required in the IND Application

The IND should include specifications with established acceptance criteria for safety testing at Phase 1. Safety testing includes tests to ensure freedom from extraneous material, adventitious agents, microbial contamination, and replication competent virus. Information on some common safety test methods is provided in more detail in section 3.2.S.4.2, Analytical Procedures.

To maximize the sensitivity of safety testing, it is important that sponsors perform each test at the stage of production at which contamination is most likely to be detected. For example, tests for mycoplasma or adventitious viruses (in vivo or in vitro) should be performed on cell culture harvest material (cells and supernatant) prior to further processing, e.g., prior to clarification, filtration, purification, and inactivation.

Additional testing will depend on the type of gene therapy product and the phase of clinical development. These tests may include assays to assess product characteristics, such as identity, purity (including endotoxin and contaminants, such as residual host cell DNA, bovine serum albumin (BSA), DNase), and potency/strength.

Sponsors should provide a description of all the analytical procedures used during manufacturing to assess the manufacturing process and product quality. In the original IND submission, descriptions should have sufficient detail so that FDA can understand and evaluate the adequacy of the procedures. FDA recommends that sponsors develop detailed SOPs for how analytical procedures are conducted at early stages of product development as a part of the sponsor's quality system. FDA acknowledges that, during product development, analytical methods may be modified to improve control and suitability. However, assay control is necessary during all phases of clinical development to ensure product quality and safety and to allow for comparability studies, following manufacturing changes.

Safety testing on the DS should include microbiological testing, such as bioburden (or sterility, as appropriate), mycoplasma, and adventitious viral agent testing, to ensure product quality. Guidelines and/or procedures for many safety tests have been described in detail, elsewhere (e.g., bioburden [1] sterility [2] mycoplasma [3], adventitious agent testing, and tests for specific pathogens [4]).

Sponsors should list DP specifications in the original IND submission. The testing plan should be adequate to describe the physical, chemical, or biological characteristics of the DP necessary to ensure that the DP meets acceptable limits for identity, strength (potency), quality, and purity. Product lots that fail to meet specifications should not be used in clinical investigation without FDA approval. For early phase clinical studies, FDA recommends that assays be in place to assess safety (which includes tests to ensure freedom from extraneous material, adventitious agents, and microbial contamination) and dose (e.g., vector genomes, vector particles, or genetically modified cells) of the product.

Sponsors should describe the analytical procedures used for testing the DP. If the analytical procedures are the same as those for the DS, sponsors do not need to repeat this information unless there is a matrix effect from the DP on assay performance.

Product Release Testing

FDA recommends that product release assays be performed at the manufacturing step at which they are necessary and appropriate. For example, mycoplasma and adventitious agents release testing is recommended on cell culture harvest material. In addition, sterility, endotoxin, and identity testing are recommended on the final container product to ensure absence of microbial contamination or to detect product mix-ups that might have occurred during the final DP manufacturing steps (e.g., buffer exchange, dilution, or finish and fill steps).

If a DP is frozen before use, FDA recommends that sponsors perform sterility testing on the product prior to cryopreservation so that results will be available before the product is administered to a patient. However, if the product undergoes manipulation after thawing (e.g., washing, culturing), particularly if procedures are performed in an open system, sterility testing may need to be repeated.

FDA also recommends that the results of in-process sterility testing be incorporated into the acceptance criteria for final product specifications.

Alternative Microbiological Methods and Rapid Sterility Tests

Analytical procedures different than those outlined in the United States Pharmacopeia (USP), FDA guidance, or Code of Federal Regulations (CFR) may be acceptable under IND if sponsors provide adequate information on test specificity, sensitivity, and robustness. Examples of alternative methods, which may be needed for live cells, include rapid sterility tests, rapid mycoplasma tests (including PCR-based tests), and rapid endotoxin tests. FDA recommends that sponsors plan to demonstrate equal or greater assurance of the test methodology, compared to a compendial method, prior to licensure, as required under 21 CFR 610.9.

For reference, Sec. 610.9, Equivalent methods and processes, states the following:

Modification of any particular test method or manufacturing process or the conditions under which it is conducted as required in this part or in the additional standards for specific biological products in parts 620 through 680 of this chapter shall be permitted only under the following conditions:

(a) The applicant presents evidence, in the form of a license application, or a supplement to the application submitted in accordance with 601.12(b) or (c), demonstrating that the modification will provide assurances of the safety, purity, potency, and effectiveness of the biological product equal to or greater than the assurances provided by the method or process specified in the general standards or additional standards for the biological product; and

(b) Approval of the modification is received in writing from the Director, Center for Biologics Evaluation and Research or the Director, Center for Drug Evaluation and Research.

FDA recognizes that the compendial sterility test may not be suitable for all products. For example, rapid sterility tests may be needed for ex vivo genetically modified cells administered fresh or with limited hold time between final formulation and patient administration.

For ex vivo genetically modified cells that are administered immediately after manufacturing, in-process sterility testing on sample taken 48 to 72 hours prior to final harvest is recommended for product release. For such products, aside from an in-process sterility test, we also recommend that sponsors perform a rapid microbial detection test, such as a Gram stain, on the final formulated product and a sterility test, compliant with 21 CFR 610.12, on the final formulated product.

Under this approach, the release criteria for sterility would be based on a negative result of the Gram stain and a no-growth result from the 48 to 72 hour in-process sterility test. Although the results of the sterility culture performed on the final product will not be available for product release, this testing will provide useful data. A negative result will provide assurance that an aseptic technique was maintained. A positive result will provide information for the medical management of the subject and trigger an investigation of the cause of the sterility failure. The sterility culture on the final formulated product should be continued for the full duration (usually 14 days) to obtain the final sterility test result, even after the product has been administered to the patient.

In all cases where product release is prior to obtaining results from a full 14-day sterility test, the investigational plan should address the actions to be taken in the event that the 14-day sterility test is determined to be positive after the product is administered to a subject. Sponsors should report the sterility failure to both the clinical investigator and FDA. FDA recommends that sponsors include results of the investigation of cause and any corrective actions in an information amendment submitted to the IND within 30 calendar days after initial receipt of the positive culture test result.

In addition, be aware that a product may sometimes interfere with the results of sterility testing. For example, a product component or manufacturing impurities (e.g., antibiotics) may have mycotoxic or anti-bacterial properties. Therefore, FDA recommends that sponsors assess the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71>, Sterility Tests.

DISCUSSION

The draft guidance is a welcome indication that the FDA accepts and encourages the use of alternative and rapid microbiological methods, specifically for sterility testing of short-lived products, such as advanced therapy medicinal products (ATMP; gene and cell therapy). It is appropriate that the guidance document aligns fairly well with 21 CFR 610.12; however, there are some areas that require further clarification.

For example, the use of the 19th Century Gram Stain should be viewed only as a measure of gross contamination because low levels of microbial contaminants would never be observed from a loopful of finished product.

Next, a finished product sterility test that is compliant with 21 CFR 610.12 does not require a 14 day incubation via the compendial USP Sterility Test.

In fact, 21 CFR 610.12 states the following:

Advances in technology in recent years have allowed the development of new sterility test methods that yield accurate and reliable test results in less time and with less operator intervention than the currently prescribed methods. Some examples of novel methods include the Adenosine Triphosphate (ATP) bioluminescence, chemiluminescence, and carbon dioxide head space measurement. Manufacturers may benefit from using such sterility test methods with rapid and advanced detection capabilities. Accordingly, we have amended § 610.12 to promote improvement and innovation in the development of sterility test methods, to address the challenges of novel products that may be introduced to the market in the future, and to potentially enhance sterility testing of currently approved products. This final rule provides manufacturers the flexibility to take advantage of methods as they become available, provided that these methods meet certain criteria.

Because some of these new technologies do not reply on microbial growth, or more raid growth-based methods may be validated to provide an equivalent sterility test result in less than 14 days, the full USP <71> incubation period may not be necessary.  

The teachings in 21 CFR 610.12 do allow for the sterility testing of material other than the finished drug product in its final container. The May 3, 2012 Federal Register Vol. 77, No. 86, Page 26162 – 26175 provides additional guidance on what alternative material may be comprised of, e.g., bulk material or active pharmaceutical ingredient (API), in- process material, stock concentrate material), as appropriate, and as approved in the biologics license application (BLA) or BLA supplement.

Additional guidance was provided in the same CFR reference (see page 26165 in the CFR):

As discussed in the preamble to the proposed rule (76 FR 36019 at 36021), certain allergenic and cell and gene therapy products may need to be tested for sterility at an in- process stage or some other stage of the manufacturing process (e.g., intermediate, API, bulk drug substance) instead of the final container material because the final container material may interfere with the sterility test. Likewise, as discussed in the preamble to the proposed rule, some cell therapy products and cell-based gene therapy products may need to be tested for sterility at an in-process stage or some other stage of manufacturing process because low production volumes may result in an insufficient final container material sample for sterility testing or a short product shelf-life may necessitate administration of the final product to a patient before sterility test results on the final container material are available.

Therefore, based on 21 CFR 610.12, it may be appropriate to test an alternative material and not necessarily the finished product, as long as this strategy is justified and acceptable to FDA. For these reasons, the draft guidance should be clearer on whether a sterility test on the final formulated product is a recommendation or an expectation.

Furthermore, the statement regarding demonstrating the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71>, should be extended to alternative or rapid sterility tests. These studies are, in fact, an expected part of the qualification of the alternative method and is usually conducted during the Method Suitability phase of the validation process.

Overall, the draft guidance is further assurance from the FDA that rapid and alternative methods are encouraged for use, especially on product that is short-lived or needs to be administered prior to obtaining results from the compendial sterility test. Although the document is specifically focused on gene therapy products in support of IND applications, the recommendations can be used for the routine release of the same or similar ATMPs.

Training on Rapid Sterility Testing of Gene and Cell Therapy Products

I will be speaking on this same topic during the PDA Europe Conference on Pharmaceutical Microbiology, which will be held in Berlin, Germany on October 15-16. More importantly, I will provide a comprehensive overview of rapid methods, including validation strategies, technology reviews, regulatory acceptance and applications during a two-day training course immediately following the conference on October 17-18. For more information, please visit  https://www.pda.org/global-event-calendar/event-detail/rapid-microbiology-methods.

References

1. USP<61> describes membrane filtration, plate count, and most probable number methods that can be done to quantitatively determine the bioburden of non-sterile DPs. Although 21 CFR 211.110(a)(6) does not specify a test method, it requires that bioburden in-process testing be conducted pursuant to written procedures during the manufacturing process of DPs.
2. Sterility testing may be performed on the DS when it cannot be performed on the DP, as outlined in the final rule: Amendments to Sterility Test Requirements for Biological Products (May 3, 2012; 77 FR 26162 at 26165). Sterility tests are described in 21 CFR 610.12 and USP<71> Sterility Tests.
3. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, July 1993.
4. Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications, February 2010.

CRISPR Used for Infectious Disease Diagnostics

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) are short, repeated DNA sequences found in the genomes of bacteria and other microorganisms. These sequences help to fight off bacteriophages (bacterial viruses) by slicing the invading viruses, thereby preventing the virus from replicating.

CRISPR is now being used for a variety of applications. For example, CRISPR can facilitate the replacement of a mutant gene with the correct sequence, thereby curing a genetic disorder. CRISPR is now being explored to modify antibiotics that are specific for pathogenic bacteria without impacting good bacteria that the body requires.

The Harvard Graduate School of Arts and Sciences has developed an easy to understand website for reviewing CRISPR. It includes an illustration of the basic principles of CRISPR, shown below (click for a larger image):

Science Magazine has recently published an article on how CRISPR can be used for infectious disease diagnostics. Initial areas of focus target Zika, Dengue and human papillomavirus.

Different diagnostic methods are being developed which exploit the power of CRISPR technology. For example, double stranded (ds) DNA may be extracted from a sample and amplified using isothermal preamplification by recombinase polymerase amplification (RPA). The target sDNA amplicons are then sequence specifically cleaved by a Cas12a-crRNA protein complex, which activates nonspecific cleavage of single stranded (ss) DNA. A fluorescent dye binds to the ssDNA to create a detection signal. This process is known as DNA endonuclease-targeted CRISPR trans reporter (DETECTR).

HUDSON (heating unextracted diagnostic samples to obliterate nucleases) is a method by which heat and chemicals inactivate ribonucleases (RNases) and lyses viral particles, thereby releasing nucleic acids into solution. This can be combined with SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), a Cas13-based nucleic acid detection method, to deliver a rapid (1-2 hour) diagnostic test. Using fluorescent readouts, the test can give a positive-negative readout on a paper test-strip. Read more about each of these methods, which are summarized in the illustration below (click for a larger image) with accompanying references, in Science Magazine.

Microbiology is Recognized with New Emojis

The Unicode Consortium today announced it has finalized a new set of 157 emoji. This brings the total number of approved emojis to 2,823.

The new Emoji 11.0 set is fixed and final, and includes the data needed for vendors to begin working on their emoji fonts and code ahead of the release of Unicode 11.0, scheduled for June 2018. The new emoji typically start showing up on mobile phones in August or September.

So, what does this have to do with microbiology or rapid methods? Believe it or not, there will be six (6) brand new laboratory and microbiology-related emojis including a lab coat, goggles, Petri dish, test tube, DNA molecule and finally, a microbe (which looks like E. coli on steroids!). These will be great additions to the microscope emoji which has been quite lonely for all these years!

Here is a sneak peek at the new emojis coming later this year. Please click on the image for a larger view.

Biosensors Detect Harmful Bugs in the Lungs of Cystic Fibrosis Patients

Pseudomonas aeruginosa are strains of bacteria that are widely found in the environment. Pseudomonas is a major cause of lung infections in people with cystic fibrosis. The bacteria thrive in moist environments and equipment, such as humidifiers and catheters in hospital wards, and in kitchens, bathrooms, pools, hot tubs and sinks.

Pseudomonas infections are known as opportunistic. This means the bacteria only cause infections when a person has CF or another condition that weakens the body's immune system.

Pseudomonas is one of the most common bacteria found in people with CF. About half of all people with CF have Pseudomonas. More than 60 percent of adults with CF have Pseudomonas.

The number of people with CF with the bacteria has been decreasing, however.Once Pseudomonas is established in the airways, it's very difficult to eliminate. But aggressive treatment can delay the development of long-term infection.

The rapid detection of Pseudomonas in CF patients is also paramount in treating patients. And scientists at the Imperial College London are doing just that. The following text provides an overview of their work.

Biosensors and the detection of Pseudomonas

A team of Imperial researchers has developed a tool which 'lights up' when it detects the chemical signature of harmful bacteria in the lung.

In a clinical first, the group from the Department of Medicine used the tools, called cell-free biosensors, to test samples of sputum (phlegm) from patients with cystic fibrosis (CF).

Biosensors are based on engineered DNA circuits, designed to detect changes in their environment, such as the presence of chemicals, or changes in pH or temperature.

These tools, which harness the biological machinery inside cells, can be used to quickly spot chemical traces of active microbial colonies in samples from the lung and could help to accurately diagnose bacterial infections in vulnerable patients.

In a small, proof-of-concept study, the team found that their biosensors could accurately detect markers of Pseudomonas bacteria – a leading cause of chest infections in people with weak immune systems or chronic conditions, such as CF – and were as sensitive as existing chemical diagnostic tests but could potentially be cheaper and easier to use.

Rapid diagnostic tests

The researchers are hopeful they could eventually develop their cell-free sensors into a range of rapid diagnostic tests which could be used either at home, GP surgeries or in hospital clinics or even in remote areas of the world with limited access to hospital diagnostics, at a fraction of the price of existing tests.

Professor Paul Freemont, co-founder and co-director of The Centre for Synthetic Biology and Innovation at Imperial, said: “The driving force behind this research is to show that these tools work and could be used to detect particular diagnostic markers associated with infection.”

He added: “By applying an engineering approach to biology, these systems could be altered to sense for any microbe we choose. The possibilities for public health and cost-savings for health systems could be considerable.”

Biosensors have emerged through the growing field of synthetic biology, with scientists tweaking living cells to respond to certain conditions, such as the presence of a chemical compound.

The engineered DNA circuit: when the bacterial molecules (AHL) are present they trigger the production of a coloured protein (GFP)

At the heart of the technique are lengths of engineered DNA which, when inserted into a living cell, act as a circuit. If the right signal – such as a specific chemical compound – is present, then the circuit is switched ‘on’ and the cell produces a signal in the form of a coloured output, in this case a green fluorescent protein. If the substrate is not present, then the circuit remains ‘off’ and there is no signal.

In the latest study, published this week in the journal ACS Synthetic Biology, researchers report on using a ‘cell-free’ form of sensor to test clinical samples for the first time. Instead of being contained within the membrane of a cell, the engineered DNA circuit and cellular machinery of their sensors are free-floating in a solution.

The team engineered their sensor’s circuit to respond to a molecule produced specifically by the bacterium Pseudomonas aeruginosa. These bacteria release a chemical signature in order to communicate with bacteria around them and to sense how many of them there are. As they replicate, more cells release the signature, and so the concentration of the signal increases, giving the bacteria an idea of the state of their population.

Previous studies have revealed levels of this same P. aeruginosa signature were higher in hospitalised patients than those with stable condition.

Samples taken from the lungs of patients, either with or without P. aeruginosa infection, were screened by adding them to tiny wells containing solutions of the biosensor. After four hours the samples were tested for green fluorescent protein – a sign that the bacterial signature was present.

Analysis revealed that the cell-free biosensor was able to detect the bacterial signature with the same accuracy as existing diagnostic tests, called liquid chromatography tandem mass spectrometry (LC-MS/MS). The biosensors were able to detect the concentration of the signal within a close range to LC-MS/MS.

Taking the technology forward

Currently, a major limitation for the cell-free sensors is the amount of time taken to prepare samples before testing, with lung sputum samples needing to be ‘cleaned up’ with the removal of biological materials which could interfere with the results.

In addition, as the DNA is synthetic – engineered in the lab – it would need to be disposed of carefully, with concerns around potential to contaminate the environment or pass to other organisms.

However, according to the researchers, the approach could offer significant cost-savings compared to the existing LC-MS/MS detection method. If suitably scaled up, using similar cell free biosensors could work out to be a fraction of the cost per sample.

Professor Jane Davies of the NHLI and an honorary consultant paediatrician at the Royal Brompton Hospital said: “Pseudomonas is the major infection in patients with cystic fibrosis and is closely linked to the severe lung disease which develops over time leading to premature death.

“At the moment, we only have opportunities to detect infection when patients come to clinics, perhaps every 2-3 months. A point of care test could transform the way in which we monitor patients, allowing us to treat early and personalise therapies.”

The group is now exploring how to take the technology forward and develop working prototypes, as well as a test to detect the signal in a patient’s urine. If the biosensor solution could be freeze-dried, this could even potentially take the form of a credit card-sized paper-based test, which would be affordable and ready for use in the field.

Loren Cameron, a PhD student in Freemont’s lab and one of the study authors, said: “Now we have shown these types of biosensors can be used with clinical samples, the next step is to refine our approach. We hope that in future, tools like this could be used to help test for the presence or severity of bacterial infections.” Ms Cameron is one of five postgraduate students funded as part of the Cystic Fibrosis Trust’s Strategic Research Centre for Pseudomonas infection.

Ke Yan Wen, a graduated PhD student from Freemont’s lab and also one of the lead authors, said: "We believe that cell-free systems are a promising platform for developing biosensors, since they are easy to use, produce a rapid response, and can be stored long-term in normal conditions.”

Source: Imperial College London

My Opinions on Sage Products Rapid Method Warning Letter (and a reply to Tim Sandle's Blog)

Tim Sandle published a recent blog post concerning a (FDA) Warning Letter related to an inadequate validation of a rapid microbiological method by Sage Products Inc. Tim's post reviews the warning letter findings and poses a number of follow up questions.

My opinions are as follows. Tim's original blog post is reproduced after my opinion. Tim's blog on this topic may be viewed by clicking here.

The FDA warning letter may be viewed by clicking here.


MY OPINION

This was an unfortunate event that should, in no way, dissuade firms from implementing rapid methods. To support my opinion, I need to provide clarity around the warning letter points, which are presented below in quotation marks.

“Your firm failed to establish and document the accuracy, sensitivity, specificity, and reproducibility of its test methods. Specifically, you use the [redacted] method to screen for microbiological contamination in drugs produced entirely at your facility and those manufactured under contract. This [redacted] screening method [redacted] for microbiological examination of your liquid drug products is not adequate for its intended use. You attempted to validate your [redacted] microbial detection method, but were not able to demonstrate that it could reliably and repeatedly determine whether objectionable microorganisms were present in your drugs.”

This tells me two things. First the rapid method was not adequately validated to demonstrate equivalency to the compendial method, which is a requirement in USP and the other compendia.

“After receiving three consumer complaints for discoloration of this product, you initiated testing of your retains using both the modified U.S. Pharmacopeia (USP) microbiological limits method and the [redacted] method. Both analyses found microbial contamination. Notably, the USP modified method [redacted] found an exceedingly high microbial count of over 57,000 CFU/ml, and also identified Burkholderia cepacia, an objectionable microorganism, in this product lot.”

“Had your firm been utilizing a screening method capable of consistently detecting B. cepacia, these products may not have been released in the first instance.”

Obviously! It appears the “validated” rapid method did not detect the presence of microorganisms in the original sample (which is why it was apparently released) and as such, there was no positive response to illicit confirmatory testing of an objectionable, such as B. cepacia.

“FDA informed you that your [redacted] method [redacted] has not been adequately validated for detecting the presence of microorganisms, including the presence of B. cepacia. In a subsequent meeting on November 30, 2016, FDA advised you to use a verified compendial method for all bulk drug solutions and finished product microbiological testing until you could further assess the suitability of the [redacted] method.”

This statement could mean a number of things. An alternative method, including a rapid method, must be equivalent to the compendial method. For nonsterile drugs, which I assume is the case with this warning letter, you must demonstrate the finished product is within aa appropriate quantitative specification (the reason why we perform USP 61) and does not contain any objectionable or specified microorganisms (which is why we perform USP 62 and additional tests for organisms that may not be specifically covered in the compendia). Some of the reasons why the rapid method could not detect microorganisms, including B. cepacia, could be fond in the next FDA statement.

“It specifies a [redacted] dilution factor. USP <62> requires a 1:10 dilution factor. Your dilution factor is [redacted] times greater than the USP method and provides insufficient detectability to rule out the presence of objectionable microorganisms and unacceptable total counts.”

Well, this is an issue regardless of whether the method used was USP 62 or an alternative method. Diluting out the test sample more than what is prescribed in the compendia may dilute out organisms that could be present. This was a mistake that should have been avoided.

Furthermore the rapid method it not account for the enrichment step called for in USP <62>. Also “it does not include the scraping step during sample preparation, which your submitted laboratory data indicates is required to validate organism recovery.”

Again, the same sample preparation steps must be performed in an alternative method, unless you can show a different procedure gives equivalent results to the compendial or original sampling and sample preparation strategy.

There was also a comment about the use of stressed organism as part of method validation (a controversial point within pharmaceutical microbiology): “it lacks evidence that small numbers of various microorganisms, including those that are injured and stressed, can be reliably recovered. Specifically, sample effect (defined by your firm as the inhibitory effect of a sample on the growth of various microorganisms) data for B. cepacia was collected using a fresh-grown culture, not a stressed organism.”

This was an interesting finding. The use of stressed organisms fueling the validation of rapid methods has been suggesting in PDA Technical Report #33 (TR33) and up until recently, the prior version of Ph. Eur. 5.1.6. Obviously, the FDA continues to require the use of stressed organisms when validating an alternative or rapid microbiological method because organisms normal found in finished product, especially if the product is preserved, will be stressed to some extent.

The final charge was that the method validation did not “establish potential sample interference factors (e.g., enhancing or quenching) for each product formulation.”

This tells me the firm did not perform method suitability on the product to eliminate the potential for false positives and false negatives. This is a current requirement in the USP, Ph. Eur. and TR33 and if Sage did not perform these tests during their rapid method validation studies, this is a significant issue.

There was also concern that the replacement method, unlike the USP method, did “not provide for potential speciation of the detected microbial contamination in the [redacted] initial screening test.”

Some rapid methods are based on the growth of microorganisms and may provide CFUs for subsequent analysis, including confirmation of an objectionable organisms and/or microbial identification. However, other rapid methods may not be based on growth and as such, follow up analysis of positive results may not be possible unless a separate sample was tested specifically for this purpose. Because eh exact rapid method under dispute is not disclosed in the warning letter, it is not possible to conclude the capabilities of the alternative method at this time.

Tim Sandle poses the following questions in his original post on this subject, based on the Sage warning letter. I will answer each in turn:

Q. Does the validation of methods require the use “stressed” organisms?

A. Depending on the application, the use of stressed organisms should be considered. Firms do not have to test dozens of stressed organisms but should select relevant strains and use a stressing condition that is defendable. Novartis published a number of papers on stressing organisms when they validated a rapid sterility test (see the reference page at rapidmicromethods.com for downloads or links to the papers). IN any case, firms should discuss their validation plans with the relevant regulatory authority to understand if the use of stressed organisms is expected.

Q. Does validation always need to be against each product formulation (not just the strongest concentration of the most inhibitory ingredient)?

A. If method suitability testing takes into consideration the greatest (and least) challenge to the alternative method, then you may be able to justify bracketing formulations to support these studies. But this will depend on the actual formulations intended for testing. Again, discuss your strategy with the regulators up front.

Q. How are representative samples built into the validation process?

A. Samples should be selected for validation studies identical to what will be tested routinely (by the rapid method). Consideration for sample preparation, dilutions, etc. should also be noted and as required by the compendia or regulatory expectations.

Q. Does a list of objectionable organisms need to be prepared ahead of the method validation? Or is this just a B cepacia issue?

A. Firms should understand what organisms should be detected by the method, based on a product’s intended use and patient population. This should be done regardless of whether an alternative method will be validated or if the standard compendial method is utilized.

TIM'S ORIGINAL POST

In July 2017 Sage Products Inc. received a warning letter from the U.S. Food and Drug Administration (FDA). Within the warning letter was a concern about the implementation of rapid microbiological method, where the method had been used to replace a compendial method.

In the warning letter, the FDA state “Your firm failed to establish and document the accuracy, sensitivity, specificity, and reproducibility of its test methods.” This relates to the use of a rapid method.

Specifically: “You use the (b)(4) method to screen for microbiological contamination in drugs produced entirely at your facility and those manufactured under contract. This (b)(4) screening method (b)(4) for microbiological examination of your liquid drug products is not adequate for its intended use. You attempted to validate your (b)(4) microbial detection method, but were not able to demonstrate that it could reliably and repeatedly determine whether objectionable microorganisms were present in your drugs.”

The FDA are concerned about the comparative findings from the USP method and the replacement rapid method: “After receiving three consumer complaints for discoloration of this product, you initiated testing of your retains using both the modified U.S. Pharmacopeia (USP) microbiological limits method and the (b)(4) method. Both analyses found microbial contamination. Notably, the USP modified method (b)(4) found an exceedingly high microbial count of over 57,000 CFU/ml, and also identified Burkholderia cepacia, an objectionable microorganism, in this product lot.”

It seems that the firm elected to run with the results from the alternative method, despite the USP method finding an objectionable microorganism. This led the FDA to state: “Had your firm been utilizing a screening method capable of consistently detecting B. cepacia, these products may not have been released in the first instance.” Part of the reason was “because [the company] failed to include B. cepacia on the list of objectionable organisms.”

The use of the alternative method had also gone against FDA advice: “FDA informed you that your (b)(4) method (b)(4) has not been adequately validated for detecting the presence of microorganisms, including the presence of B. cepacia. In a subsequent meeting on November 30, 2016, FDA advised you to use a verified compendial method for all bulk drug solutions and finished product microbiological testing until you could further assess the suitability of the (b)(4) method.”

The concerns the FDA had with the rapid method were:

“It specifies a (b)(4) dilution factor. USP <62> requires a 1:10 dilution factor. Your dilution factor is (b)(4) times greater than the USP method and provides insufficient detectability to rule out the presence of objectionable microorganisms and unacceptable total counts.”

Furthermore the rapid method it not account for the enrichment step called for in USP <62>. Also “it does not include the scraping step during sample preparation, which your submitted laboratory data indicates is required to validate organism recovery.”

There was also a comment about the use of stressed organism as part of method validation (a controversial point within pharmaceutical microbiology): “it lacks evidence that small numbers of various microorganisms, including those that are injured and stressed, can be reliably recovered. Specifically, sample effect (defined by your firm as the inhibitory effect of a sample on the growth of various microorganisms) data for B. cepacia was collected using a fresh-grown culture, not a stressed organism.”

The final charge was that the method validation did not “establish potential sample interference factors (e.g., enhancing or quenching) for each product formulation.”

There was also criticism about the sampling and sample plan used to ensure the tested sample was representative of the lot. There was also concern that the replacement method, unlike the USP method, did “not provide for potential speciation of the detected microbial contamination in the (b)(4) initial screening test.”

The implications from the FDA letter, as well as signaling a pertinent lesson when implementing a rapid method that needs to be equivalence to or better than the rapid method, are:

• Does the validation of methods require the use “stressed” organisms?
• Does validation always need to be against each product formulation (not just the strongest concentration of the most inhibitory ingredient)?
• How are representative samples built into the validation process?
• Does a list of objectionable organisms need to be prepared ahead of the method validation? Or is this just a B cepacia issue?

If you have views on this, please add a comment.

Thanks to Nigel Halls for the heads-up about this warning letter and its considerable implications.

Longitude Prize Bacterial Dx Competition Announces Seed Funding Winners

On average antibiotics add 20 years to each person’s life. The development of antibiotics has been vital to our survival, yet the rise of antimicrobial resistance is threatening to make them ineffective in the future. The World Health Organization estimates that antibiotics treatments add an average of 20 years to all of our lives. But in the 80 years since the discovery of penicillin, our overuse of antibiotics has put pressure on bacteria to evolve resistance, leading to the emergence of untreatable superbugs that threaten the basis of modern medicine.

Clinicians often prescribe broad spectrum antibiotics to sick patients because doctors have to act quickly on imperfect information. These methods put selective pressure on microbes to evolve resistance to antibiotics. Prescriptions from clinicians are just one way in which people access antibiotics, however. There are many routes to these drugs and they vary around the world.

Radical change is needed to address the global problem of growing anti-microbial resistance, to ensure a healthcare system that can sustainably control and treat infections.

We cannot outpace microbial evolution. A new broad-spectrum antibiotic, if applied with current methods, would eventually meet new forms of resistance. The overall solution involves a long-term path towards a more intelligent use of antibiotics enabling a future of more effective prevention, targeted treatments and smart clinical decision support systems.

The Longitude Prize is a £10m prize fund that will reward a competitor that can develop a point–of–care diagnostic test that will conserve antibiotics for future generations and revolutionise the delivery of global healthcare. The test must be accurate, rapid, affordable and easy to use anywhere in the world.

Run by Nesta and supported by Innovate UK as funding partner, the Longitude Prize competition recently announced 13 winners for the second round of seed funding.

Each winning team received awards between £10,000 ($13,210) and £25,000 ($37,927) for their research proposals. Funding for the second round draws on a £250,000 grant from Merck & Company. The groups come from Australia, Belgium, India, Israel, The Netherlands, the US, and the UK.

Teams are at various stages in test developments, ranging from proof of concept to initial clinical validation to fabrication of components for prototypes. They are attempting to diagnose infections including Escherichia coli, urinary tract infections, and sepsis.

Many teams are working on technology that will detect the susceptibility of bacteria to antibiotics, which will support specific antibiotic treatments for bacterial infections and minimize the spread of drug resistance.

Here is an overview of the Discovery Award Winning Teams:

• ID Genomics, a Seattle-based company, is developing "bacterial fingerprinting" technology that will reduce prescription errors and overuse of broad-spectrum antibiotics by improving the speed and accuracy of the antibiotic prescribing process. ID's Clonet rapid diagnostic test determines the bacterial fingerprint of different strains of the main pathogens in urinary tract infections (UTIs) in less than 30 minutes, which is then matched against the company's reference database of thousands of bacterial strains and their profiles. A clinician can then prescribe the antibiotic best suited to their infection.   
• Coris BioConcept, a Belgium-based company, specializes in developing, manufacturing, and marketing rapid diagnostic tests based on strip chromatography with the use of colloidal gold particles, latex microspheres, or fluorescent dyes (ICT). The ICT range detects infectious disease including enteric and respiratory pathogens, and could potentially be used to confirm if a bacteria is resistant or sensitive to carbapenem antibiotics.  
• Drug and Diagnostics for Tropical Disease is developing a test to determine if an antibiotic can still bind to its targeted protein or not, providing an answer within minutes without specialized equipment and allowing a doctor to test a patient in the office.
• A consortium called EPDAL:Bradford and Lincoln plans to design an efficient and inexpensive diagnostic test to identify the most significant gram-negative pathogens. On the premise that protein chemistry is the same in the structure of gram-negative bacteria (GNB) or proteins, the company will exploit the ability of protein elements to select different color complexing molecules and use differential staining to organize bacteria into separate categories based on their protein elements and color complex-elution properties.
• Embryyo, an Indian company, is working on a diagnostic device that will facilitate in early detection of bacterial or viral infection on the bloodstream, at the onset of a pathogenic infection when pathogen counts are very low. Based on microfluidic cell separation and flow focusing microdevices, the device will potentially aid in the differential diagnosis of bacterial versus viral infections.
• Encompass Consortium, based in Australia, is developing a method to determine antimicrobial susceptibility with the accuracy and speed needed to influence a physician's initial choice of antibiotics. The company aims to miniaturize and automate its first-generation flow cytometry-assisted antimicrobial susceptibility tests (FAST) for near point-of-care use.
• Going Against the Flow, another Australian company, is working on a point-of-care test that will detect the activation of neutrophils to allow early detection and treatment of sepsis. Instead of detecting infection directly, the test will rely on the body's sensitive and quick response to bacterial infections using original yet simple methods to detect the early response to neutrophils.
• Microplate-Strathclyde Biomedical Engineer and SIPBS's approach will bring together expertise to develop a rapid diagnostic test for antimicrobial susceptibility.
• Module Innovations, an Indian company, is developing USense, a diagnostic platform based on color changing polymers for rapid microbial detection.
• OxTB, Cambridge and Oxford, is working on a bedside test that uses whole-genome sequencing (WGS) to determine the presence of Mycobacterium tuberculosis in a clinical sample, predict drug susceptibility, and inform disease surveillance by demonstrating the genetic relationships to previously witnessed strains.
• Prismatix, an Israeli-based company, has developed a method of phase-shift reflectometric interference spectroscopic measurements (PRISM) that monitor bacterial activity silicon-based microstructures in real time.
• RAPDIF, based out of The Netherlands, plans to improve the diagnosis of febrile diseases in sub-Saharan Africa. It aims to develop a diagnostic tool that can identify bacterial infections.
• Rapid AMR Detection Team, from the University of Bristol, is developing an optical technique to quickly detect whether bacteria are alive or not, relying on optical detection of the state of bacteria after injecting antibiotics and not relying on bacterial growth.

Source: The Longitude Prize Challenge

Inclusion of Rapid Test Results Leaves Gaps in CDC Foodborne Illness Data

For the first time, the CDC’s yearly report on foodborne illnesses in the U.S. included infections that were identified only with rapid diagnostic tests, which can speed up treatment for patients but lead to important gaps in data, researchers said.

Culture-independent diagnostic tests (CIDTs) are increasingly being used by clinicians to detect enteric infections because they produce results more quickly than traditional culture methods, researchers from the CDC and other public health facilities across the country wrote in MMWR. However, used alone, these tests tell only part of the story, leaving out important information on pathogen subtypes and antimicrobial resistance, and making it difficult to monitor trends or link infections to outbreaks, the researchers said.

Such information can be obtained only if a reflex culture is performed on the CIDT-positive specimen, the researchers said. But according to their report, which compared surveillance data for 15% of the U.S. population from 2013 to 2016, a large proportion of positive CIDT results do not receive these much-needed cultures.

“We need foodborne-illness trend data to monitor progress toward making our food supply safer,” Robert Tauxe, MD, MPH, director of the CDC’s Division of Foodborne, Waterborne, and Environmental Diseases, said in a statement. “It’s important that laboratories continue to do follow-up cultures on CIDT-positive patients so public health officials can get the information needed to protect people from foodborne illness.”

Potential for false-positive results

Including CIDT results in the CDC data for 2016 raised the incidence rates for six of the most reported bacterial foodborne illnesses. However, interpreting these increases is complicated, the researchers said.

As an example, they noted that the incidence of culture-confirmed infections with Campylobacter — the most commonly reported bacterial foodborne illness — was “significantly lower” in 2016 compared with the average over the previous 3 years. However, a “slight but not significant” increase occurred when infections that were diagnosed only through CIDTs were included.

Further, Campylobacter is one of the pathogens for which antigen-based CIDTs are often used. These tests can produce a large number of false-positive results, leading to skewed estimates of incidence, the researchers said.

“When interpreting incidence and trends in light of changing diagnostic testing, considering frequency of testing, sensitivity, and specificity of these tests is important,” they wrote. “The observed increases in incidence of confirmed or CIDT positive–only infections in 2016 compared with 2013 to 2015 could be caused by increased testing, varying test sensitivity, an actual increase in infections, or a combination of these reasons.”

FoodNet data

The researchers looked at preliminary 2016 data from the CDC’s Foodborne Disease Active Surveillance Network, also called FoodNet, which monitors cases of nine foodborne disease at 10 sites in Connecticut, Georgia, Maryland, Minnesota, New Mexico, Oregon, Tennessee and certain counties in California, Colorado and New York, and compared the incidence rates with data from 2013 to 2015.

The sites reported 24,029 foodborne infections in 2016, including 5,512 hospitalizations and 98 deaths. Campylobacter was the leading cause of bacterial foodborne illness with 8,547 reported infections, followed by Salmonella (8,172), Shigella (2,913), Shiga toxin-producing Escherichia coli (1,845), Cryptosporidium (1,816), Yersinia (302), Vibrio (252), Listeria (127) and Cyclospora (55).
Salmonella typhimurium infections, which often are linked to beef and poultry, decreased 18% in 2016 compared with the average incidence for 2013 to 2015. The reduction is possibly due to regulatory action by the USDA to reduce Salmonella contamination in poultry and vaccination of chicken flocks, according to a CDC news release.

The CDC said increases in Yersinia, Cryptosporidium, and Shiga toxin-producing E. coli infections were likely due to the increased use of CIDTs.

“This report provides important information about which foodborne germs are making people sick in the United States,” Tauxe said. “It also points out changes in the ways clinicians are testing for foodborne illness and gaps in information as a result.”

The researchers said previous analyses have indicated that the number of foodborne infections in the U.S. “far exceeds those diagnosed” and that CIDTs might be making them “more visible.” But they said more tools are needed to accurately interpret FootNet surveillance data in light of these changes to testing practices.

“FoodNet is collecting more data and developing these tools,” they wrote. “With these, FoodNet will continue to track the needed progress toward reducing foodborne illness.” – by Gerard Gallagher

Reference

Marder EP, et al. MMWR Morb Mortal Wkly Rep. 2017;doi:10.15585/mmwr.mm6615a1

New Paper on Using Statistics When Validating Rapid Microbiological Methods

From 2010 to 2013, European Pharmaceutical Review published a very successful series on rapid microbiological methods (RMM) that included such topics as how to demonstrate a rapid method is equivalent to a conventional or pharmacopoeial method. PDA Technical Report #33, the recently revised USP chapter <1223> and the proposed changes to Ph. Eur. Chapter 5.1.6 all recommend the use of statistical analysis when validating rapid methods and establishing equivalence. However, many microbiologists find the concepts of statistical analysis and hypothesis testing intimidating and, in some cases, unapproachable. Once and for all, it was time to demystify statistics and provide a framework for embracing the mathematical models required when validating novel RMM technologies. 

To accomplish this, I have teamed up with two of the industry’s leading experts on statistical analysis and the validation of rapid microbiological methods: Edwin R. van den Heuvel, PhD (Eindhoven University of Technology) and David Roesti, PhD (Novartis Pharmaceuticals).

Our paper, The Role of Statistical Analysis in Validating Rapid Microbiological Methods, has recently been published in this month’s European Pharmaceutical Review. Clicking on the publication title (above) will give you online access to the paper (this is free to view but you may need to sign up for access). Alternatively, please copy and past the following address into your web browser:

http://www.europeanpharmaceuticalreview.com/46555/european-pharmaceutical-review-magazine/past-issues/issue-6-2016/rmms-supplement/

This comprehensive article discusses the concepts of statistics and hypothesis testing, including:

• How to formulate a null and alternate hypotheses as it relates to rapid method validation 
• Identifing the appropriate statistical test
• How to calculate a p-value
• Comparing the p-value with a chosen alpha level

Next we, dive into equivalence and non-inferiority tests, which we believe is the preferred strategy for demonstrating a rapid method is as good as and/or no worse than the existing microbiological method you wish to replace. The article specifies how to select lower and upper equivalence levels and also provides case studies on how to utilize these statistical tools.

EMA Promotes the Use of Rapid Microbiological Methods for Water Testing

In 2005, the European Medicines Agency (EMA) explored the regulatory consequences of implementing an alternative method for rapid control of microbiological quality of water for injection (WFI) and Purified water. Discussions between the Quality Working Party, the Good Manufacturing Practices and Good Distribution Practices Inspectors Working Groups and the Committees for Human and Veterinary Medicinal Products concluded Since it is expected that the water will continue to meet Ph. Eur. specifications, if tested, no change to dossier requirements* (variations) would be involved and therefore no regulatory impact on individual products would normally be anticipated.

• *However, this will depend on the level of detail in the original dossiers concerned.

Following discussions over the last 2-3 years around the revision of the European Pharmacopoeia (Ph. Eur.) WFI monograph (0169), the Water Working Party concluded that there was evidence to support a revision of the monograph, which proposes to take account of current manufacturing practices using methods other than distillation for producing water of injectable quality.

The Ph.Eur. monograph (Monograph 169) was revised to include, in addition to distillation, reverse
osmosis (RO) coupled with suitable techniques, for the production of WFI.

In order to provide clarification and guidance in relation to the use of reverse osmosis in the manufacture of WFI and also to provide more detailed guidance on the control of biofilms, the Water Working Party developed a draft set of Questions and Answers that were sent out for public consultation on August 5, 2016, with an end of consultation date (i.e., the deadline for comments) of November 4, 2016. The draft Q&A is intended to provide preliminary guidance until such time the on-going revision of Annex I of the GMP guide is complete.

One of the questions posed by the working party is, “What testing should be employed during initial qualification and routine operation sampling?”

In answering this question, the working party states:

• Use of rapid microbiological methods should be employed as a prerequisite to the control strategy to aid with rapid responses to deterioration of the system.” 

• Methods to be considered should include, “rapid endotoxin testing – use of more sensitive and point of use test methods,” and “quantitative microbiological test methods – in line with Ph.Eur. 5.1.6 monograph Alternative Methods for control of Microbiological Quality. 

• Due consideration should be given to employing alternate methods for the rapid quantitative determination of the contamination levels existing within the water system. The validation of such system should be in line with the above referenced monograph.

As such, EMA is encouraging the industry to employ rapid microbiological methods for the quantitative assessment of viable organisms and the presence of endotoxin in WFI and purified water. Additionally, the use of such methods should be validating according to Ph. Eur. chapter 5.1.6.

In my opinion, this is long-overdo step in the right direction. Technologies for the rapid analysis of pharmaceutical grade water have been available for years, and a number of firms who have experienced water system-related contamination events could have benefited from using a rapid method to quickly detect, and most importantly, remediate issues that have directly impacted their manufacturing facilities and/or finished product.

The full Q&A draft may be viewed at:

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/08/WC500211657.pdf.

Comments on the draft are due by November 4, 2016.