Sunday, 18 March 2018

Outbreak of fungal central nervous system and osteoarticular infections

A new research article of interest:

A multistate outbreak of fungal central nervous system (CNS) infection and septic arthritis was detected in the United States in late September 2012. Over 700 patients who received epidural injections of methylprednisolone produced at a single compounding center (New England Compounding Center) developed meningitis with or without posterior circulation stroke and/or spinal or paraspinal infections, and more than 30 patients who received intraarticular injections of the same drug developed osteoarticular infections [1-3]. Exserohilum spp, a dematiaceous (brown-black) fungus, has been the most commonly identified fungus (picture 1).

The United States Centers for Disease Control and Prevention (CDC) issued recommendations for the diagnosis and management of patients affected by the outbreak. These recommendations can be found on the CDC website.

This outbreak highlights the importance of suspecting environmental contaminants as a cause of infection introduced by a potentially contaminated drug or other compound in patients with a suggestive clinical presentation. This is particularly relevant in patients who have received products prepared by compounding pharmacies, which have substantially less federal oversight than traditional pharmaceutical companies. (See 'Compounding pharmacies'below.)

The epidemiology, clinical manifestations, and diagnosis of fungal CNS and osteoarticular infections associated with the outbreak will be discussed here; the treatment of such infections is presented separately. Infections due to dematiaceous fungi and Aspergillus spp that are not associated with the outbreak are also discussed separately. (See "Outbreak of fungal central nervous system and osteoarticular infections in the United States: Treatment" and "Central nervous system infections due to dematiaceous fungi (cerebral phaeohyphomycosis)" and "Epidemiology and clinical manifestations of invasive aspergillosis", section on 'Central nervous system infection' and "Diagnosis of invasive aspergillosis".)

Posted by Dr. Tim Sandle

Saturday, 17 March 2018


A new article of interest:

Beyond the usual engineering aspects of validation with regard to specification, installation, commissioning, qualification, and calibration, the two important aspects of validation of dry heat processes are thermal validation and endotoxin validation. Sterility is the most important and absolutely essential characteristic of a parenteral product. Sterility means the complete absence of all viable microorganisms. Depyrogenation devices, such as tunnels, are used in the pharmaceutical industry to prepare components for aseptic filling. To qualify such devices, various pharmacopoeias require depyrogenation devices to be periodically challenged with high levels of bacterial endotoxin. Although the pharmacopoeias state the acceptance criteria, little consideration is given to the practical approach. The review highlight the theoretical concept of depyrogenation and the various tests performed for the qualification of Depyrogenation Tunnels.

See: Depyrogenation article

The article cites: Tim Sandle, A Practical Approach to Depyrogenation Studies Using Bacterial Endotoxin.; J GXP Compli. 2011; 15(4): 90-96

Posted by Dr. Tim Sandle

Friday, 16 March 2018

An unusual form of antibiotic resistance in pandemic cholera

Researchers have now shown that the enzyme that makes the El Tor family of V. cholerae resistant to those antibiotics has a different mechanism of action from any comparable proteins observed in bacteria so far. Understanding that mechanism better equips researchers to overcome the challenge it presents in a world with increasing antibiotic resistance.

Cationic antimicrobial peptides, or CAMPs, are naturally produced by bacteria and by animals' innate immune systems and are also synthesized for use as last-line drugs. Cholera strains achieve resistance to CAMPs by chemically disguising the bacterium's cell wall, preventing CAMPs from binding, disrupting the wall and killing the bacterium.

Researchers had previously had shown that a group of three proteins carried out this modification and elucidated the functions of two of the proteins. Scientists reported the role of the third protein -- the missing piece in understanding CAMP resistance -- in a new paper.

Lipid A modification is a defense mechanism observed in other bacteria, but detailed biochemical characterization of AlmG showed that the way this process occurred in cholera was unique.


Jeremy C. Henderson, Carmen M. Herrera, M. Stephen Trent. AlmG, responsible for polymyxin resistance in pandemicVibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferasesJournal of Biological Chemistry, 2017; 292 (51): 21205 DOI: 10.1074/jbc.RA117.000131

Posted by Dr. Tim Sandle

Thursday, 15 March 2018

New Guidance on GxP data integrity

The U.K. Medicines Healthcare products Regulatory Agency (MHRA) has published its new guidance on data integrity. It is titled "‘GXP’ Data Integrity Guidance and Definitions."

This document provides guidance on the data integrity expectations that should be considered by organisations involved in any aspect of the pharmaceutical lifecycle or GLP studies regulated by MHRA.

The way regulatory data is generated has continued to evolve in line with the ongoing development of supporting technologies such as the increasing use of electronic data capture, automation of systems and use of remote technologies; and the increased complexity of supply chains and ways of working, for example, via third party service providers. Systems to support these ways of working can range from manual processes with paper records to the use of fully computerised systems. The main purpose of the regulatory requirements remains the same, i.e. having confidence in the quality and the integrity of the data generated (to ensure patient safety and quality of products) and being able to reconstruct activities.

According to Tim Sandle, in his paper on the subject in relation to microbiology: "Data integrity refers to maintaining and assuring the accuracy and consistency of data over its entire life-cycle, and is a critical aspect to the design, implementation and usage of any system which stores, processes, or retrieves data. Data integrity is a key regulatory concern and guidance documents have been produced by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Moreover, data integrity in relation to the microbiology laboratory is featured in several FDA warning letters, especially in relation to sample handling and reading."

The new guidance is intended to be a useful resource on the core elements of a compliant data governance system across all GxP sectors (good laboratory practice, good clinical practice, good manufacturing practice, good distribution practice and good pharmacovigilance practice).

The aim is to address fundamental failures identified by MHRA and international regulatory partners during GLP, GCP, GMP and GDP inspections; many of which have resulted in regulatory action.

See the UK Government website, to access the document.

Posted by Dr. Tim Sandle

Low-cost tool for detecting bacteria in water

Food scientists have developed a new, rapid and low-cost method for detecting bacteria in water or a food sample. Once commercially available, it should be useful to cooks using fresh fruits and vegetables, for example, and aid workers in the field responding to natural disasters.

The method is a sensitive and reliable bacteria-detecting chip that can test whether fresh spinach or apple juice, for example, carry a bacterial load. The chip, used with a light microscope for optical detection, relies on a "capture molecule," 3-mercaptophenylboronic acid (3-MBPA) that attracts and binds to any bacteria. The chemical detection method, "surface-enhanced Raman spectroscopy" (SERS), relies on silver nanoparticles. The techniques are now in the patenting process.


Brooke Pearson, Alexander Mills, Madeline Tucker, Siyue Gao, Lynne McLandsborough, Lili He. Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matricesFood Microbiology, 2018; 72: 89 DOI: 10.1016/

Posted by Dr. Tim Sandle

Wednesday, 14 March 2018

Epigenetics Includes Process that Alter Gene Activities

Since ancient times, we’ve been told that our genes can’t be changed and it’s our destiny too. But surprising today, it’s said that it can be changed by our lifestyle, habits, and even finances. What does it mean for our kids?

A guest post by Krutika Bhoot

Epigenetics heralded as an important biological discover after DNA, it is a new branch associated with genetics. Until now, we're made believe that we can't alter our genes with which we were born. A new branch of genetics discovered it that our genes get turned on and off and also expresses to lesser or greater degrees based on some important factors such as lifestyle. Let's have a look how epigenetics functions and how can we improve our lives with it.

What is Epigenetics?

The ‘Epi’ in epigenetics word is derived from a Greek word meaning “over” or “above”. It is demarcated as the study of any process that changes our gene activity with making no alteration in the DNA sequence. In simple words; it’s basically the study of gene expression – where you can see how external factors influence our genes to go turn on and turn off, also up and down.

A project named ‘the Human Genome’ recognized approximately 25,000 genes in the DNA of the human. Deoxyribonucleic acid (DNA) is universally regarded as the instructions code that the body utilizes to build and reproduce itself.  Genes themselves also requires instructions for what to do and when and where to do it.

Epigenetic modification known as tags offers the instructions. Many of these tags got discovered for human benefits. But it classifies in two chief groups such as methyl group and second histones. The methyl group is made of carbon and hydrogen while on the other hand, the histones, basically is a kind of protein. To know how exactly tags functions, suppose a gene as a lamp. The first group (Methyl) acts as an on and off switch that actually turns a gene on or off. While Histone performs like a dimmer switch that regulates gene activity up or down. It’s been revealed that we’ve 4 million of these switches that are triggered by factors like our environment and lifestyle.

Clues from Identical Twins:
A research was made on identical twins, who possessed similar genetic material, has offered scientists a unique window to find some epigenetic changes. The effect of environment and lifestyle factor on genes is so powerful that genes of twins can deviate expressively during their lives. The study unveils that identical twin that lives apart most of the time, their genes after a time becomes less alike. 

You might expect both the twins may have same health histories but still, they show different incidences of many diseases such as mental disorders like Alzheimer’s, alcoholism, schizophrenia and bipolar disorder and talking about the physical disorders like cancer, diabetes, RA (rheumatoid arthritis) and Crohn’s disease. It is because of epigenetic drift - an alteration in gene expression over a time.

Lifestyle Factor for Genes:

A professor named Dr. Rudolph Tanzi working at the department of neurology at Harvard University Medical School. He is one of those researchers who co-discovered three genes that causes early arrival familial Alzheimer’s disease. Along with a global leader (Dr. Deepak Chopra) in the arena of mind-body medicine, he also co-authored a book “Super Genes: Unlock the Astonishing Power of Your DNA for Optimum Health and Well-Being”.

In their book, they talked about a disease-related gene that it's only 5% disease-associated gene mutations that are utterly deterministic else 95% can be certainly influenced by our diet, behavior and other environmental conditions. The present model of well-being generally ignores gene, but some studies have revealed that a program of positive lifestyle alters 4 to 5 thousand different gene activities.  According to Chopra and Tanzi; you are not simply the complete packet of total genes you were born with but it’s you who is the user and controller of your genes and in simple words, you’re the only author of your story.

It's exciting news because it means you have a great deal to control your health and your future. You are definitely not at the mercy of your genetic material only that you got at your birth.

Gene Expression Modulates based on Diet, Sleep, and Exercise:

Diet indeed plays a significant role in affecting the health of your DNA. A rich diet of refined carbohydrates that increases high blood glucose damages your DNA. While on the other side of the coin, compounds like sulforaphane (found in broccoli, cauliflower, Kale, and Cabbage, etc.), epigallocatechin gallate (green tea), resveratrol (red wine) and curcumin (turmeric) can slow or possibly reverse DNA damage.

Sleeping less to complete your projects? Or not sleeping for few days due to exams? Well, as per a research inadequate sleep is also one of the reasons for disrupting genetic activity. A team of scientists examined how sleep influences your gene function and they found that insufficient sleep for just a single week can alter the activity of over 700 genes.

Not surprising, the physical exercise is well accepted for topping the best things one can do to improve overall health (physical and mental well-being). Now one can easily find several pieces of evidence that physical exercise positively affects our gene expression. As per the latest study of the brains of old mice examined 117 genes that were expressed contrarily in animals’ brain that ran frequently, associated to those that were inactive.

So Do Stress, Our Relationships, And Thoughts:

One can notice how tangible factors such as sleep, exercise, and diet affect our genes but it's also not that difficult to observe how intangibles like our relationships with people, stress and our thoughts affect our genes. Mindfulness Meditation is considered to be the best stress reducing techniques turning down the expression of our pro-inflammatory genes. Chronic inflammation includes 7 top leading causes of death such as heart disease, diabetes, cancer and Alzheimer’s.

You may think that you would have to meditate for many years to alter gene expression properly but some measurable changes can be observed even in eight hours of meditation. However, remarkable effects were found in experienced meditators in comparison with new to the practice.

Epigenetics Changes last for Several Generations:

Epigenetics is indeed one of the most amazing discoveries because the epigenetic changes generally don’t stop with you only. These epigenetic signals from our environment pass from one generation to other and sometimes for many generations with no changes in gene sequence. 

Most U.S. electricity demand can be met by renewables

A new study shows that up to 80 percent of the U.S. business and home energy demands can be met through the use of renewable energy sources, especially wind and solar power. The main limitation is with energy storage.

In fact, it is theoretically possible to meet the entire U.S. energy demand through renewable sources. However, the 100 percent aspirational target in terms of electrical demand would require huge investment in investment in greater storage capacity and power transmission capabilities. Efficient storage solutions are essential with renewable sources due to the cycle of natural variability.

The assessment of energy comes from a collaborative effort from the University of California, Irvine; the California Institute of Technology; and the Carnegie Institution for Science. The group of scientists analyzed thirty-six years of hourly U.S. weather data (collected during the period 1980 to 2015). This analysis allowed the researchers to understand the primary geophysical barriers in place that would affect supplying electricity to U.S. homes and businesses only using solar and wind energy sources.

One of the researchers, Professor Steven Davis explains to Laboratory Manager magazine: “We looked at the variability of solar and wind energy over both time and space and compared that to U.S. electricity demand.”

He proceeds to outline: “What we found is that we could reliably get around 80 percent of our electricity from these sources by building either a continental-scale transmission network or facilities that could store 12 hours' worth of the nation's electricity demand."

The researchers said that such expansion of transmission or storage capabilities would mean very substantial—but not inconceivable—investments. They estimated that the cost of the new transmission lines required, for example, could be hundreds of billions of dollars. In comparison, storing that much electricity with today's cheapest batteries would likely cost more than a trillion dollars, although prices are falling.

Posted by Dr. Tim Sandle

Premature births linked to changes in mother's bacteria

Changes to the communities of microbes living in the reproductive tract of pregnant women could help to spot women at risk of giving birth prematurely, according to Imperial College London researchers.

A study of hundreds of women, carried out at Imperial College London, found that subtle changes to the bacteria present in the vagina were strongly associated with the mother's waters breaking early and preterm birth -- the baby being born before 37 weeks.

According to the researchers, the findings show that a shift away from the usual healthy balance of vaginal bacteria was associated with waters breaking early, and could have an impact on the health of mother and baby, including increasing the risk of sepsis for newborns.

Samples were collected from a prospective group of 250 pregnant women with and without risk factors for giving birth prematurely -- such as having a history of preterm birth or miscarriage -- of which 27 did in fact have a premature birth. They also collected samples from a second, smaller group of 87 women who presented to hospital with premature membrane rupture. All patients were seen at Queen Charlotte's and Chelsea Hospital and Chelsea and Westminster Hospital.

Previous research has shown that over the course of pregnancy the bacteria that colonise the vagina become less diverse and are dominated chiefly by Lactobacillus species, the same type of bacteria found elsewhere in the body including the gut and mouth.

Analysis of the team's samples revealed that premature membrane rupture was associated with a shift in microbiota, with a drop in Lactobacillus and an increase in other types of bacteria, including potentially harmful bugs such as Staphylococcus and Streptococcus.

The team also analysed samples from the small group of women with premature rupture before and after the preventative antibiotic treatment -- oral erythromycin, four times a day for 10 days. Swabs were taken before treatment and then at 48 hours, one week and two weeks.

For those women whose microbial makeup was dominated by Lactobacillus before the treatment, the antibiotics resulted in a decline in Lactobacillus and a greater diversity of bugs. However, in those women with reduced Lactobacillus to begin with, the treatment was beneficial in some, reducing the amount of potentially harmful bacteria as well.


Richard G. Brown, Julian R. Marchesi, Yun S. Lee, Ann Smith, Benjamin Lehne, Lindsay M. Kindinger, Vasso Terzidou, Elaine Holmes, Jeremy K. Nicholson, Phillip R. Bennett, David A. MacIntyre. Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycinBMC Medicine, 2018; 16 (1) DOI: 10.1186/s12916-017-0999-x

Posted by Dr. Tim Sandle

Tuesday, 13 March 2018

High-resolution imaging gives new view of how fungi grow

Many fungal species grow through a process of vesicle secretion that can be applied in a biotechnology setting to make commercial or medical products. However, the details of this process are unclear. Researchers in Japan used a high-speed imaging technique to visualize hyphal growth in the fungus Aspergillus nidulans. Several new features were uncovered, including the discovery that different vesicle types move at different velocities.

Most fungi grow through the extension of hyphae, which are fiber-like structures made of one or more cells encased within a single, long cell wall. The growing tip of a hypha is loaded with ChsB, a cell wall synthesis enzyme that helps to lengthen the cell wall. ChsB is carried through the hypha by small cellular sacs called vesicles. In order for hyphae to grow, the transport and release of ChsB by these vesicles needs to be precisely timed-and exactly how this timing is achieved is unclear.

To address these shortcomings, the researchers used a technique called high-speed pulse-chase imaging, which allowed them to follow ChsB by fusing it to a marker emitting red fluorescent light. After bleaching away background red light using a laser beam, a separate beam was aimed at a single spot in the hypha, causing ChsB to fluoresce. The team was then able to trace the movement of the fluorescent ChsB as vesicles carried it around the hypha.

The technique resulted in minimal background and allowed images to be taken, on average, every 50 milliseconds-a level of precision not previously seen in hyphae. Correspondingly, the study yielded many new insights into the timing of vesicle movement during fungal growth.


Lu Zhou, Minoas Evangelinos, Valentin Wernet, Antonia F. Eckert, Yuji Ishitsuka, Reinhard Fischer, G. Ulrich Nienhaus, and Norio Takeshita. Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cellsScience Advances, 2018; DOI: 10.1126/sciadv.1701798

Posted by Dr. Tim Sandle

Monday, 12 March 2018

Comparing between Laboratory Fume Hoods (Ducted and Ductless Hoods)

No doubt that fume hood is a major investment and it is better to clear you mind with the options that are actually available. There are many factors that need to be considered to make sure that hood which you select for the lab is a perfect fit for the application. It does not matter whether it is for the up front, filter maintenance or the makeup air requirements. The investment that is being made first should be cleared and the decision on whether the option that you have chosen is actually worth or not must be taken. After all, it is an investment and of course it needs to be quite worth or not.

A guest post by Naveen Kumar

Things you need to know about the Chemicals that are being used:

Now that you have made up your mind that you wish to go ahead and choose the effective option of Laboratory Fume Hoods (Ducted and Ductless Hoods), you might get confused on which is the right option. At such time, understand that your hood must be considered only when you take into account all sorts of requirements and effective features. This would make sure that the chemicals which you would be using in the hood are best suitable for the filtration. Talking about the chemicals, make it clear on which all chemicals you would be using and whether it can actually effectively get filtered or not.

How often the filters need to be replaced:

Whether you are looking for the ductless hood option or the one with the duct, you need to understand if you require filter section or not. Choosing the ductless hood option would save a lot of money when it gets heated and cooled down at the building price. Besides, there is also the replacement filter price to be considered to make sure it fits in your budget. It is your filter vendor who would be able to help you understand the right way. Besides, your filter also gets created in much better manner. To make sure the filter works with better life, you need to replace it in every 12 months.

Lab Renovation matters equally:

In case, you are planning to renovate your lab then considering between the Laboratory Fume Hoods (Ducted and Ductless Hoods) can be quite a confusing task. Your building space may now allow the HVAC alternation. That is when it is important to go ahead and choose the ductless hood. This way, it becomes quite a convenient option for you to go in the lab when there is a electrical outlet needed.

Other than this, you need to also consider whether you are planning to be a greener lab. This means, the filters that needs to be sued shall be recyclable. The best part about ducted option is it can handle the most convenient chemical applications and has the large volumes, where as in ductless fume and Laminar Flow Hoods, it is easy to install and extremely mobile option that you can choose.  In most of the hood option, you can also choose the plastic concept for which you would have to know which the right fit for the lab is.

Liquid Immersion Microbial Challenge Tests

The container closure system for pharmaceutical products intended to be sterile is critical and this criticality relates to the physical properties of the container closure system in that a poorly designed or manufactured system will result in microbiological penetration. Factors to take into account with, for example, rubber-stoppered glass vials include having the correct dimensional specifications for the internal diameter of the neck opening and its depth, the internal and external diameters of the flange. Other factors are the concentricity of the flange, the neck and the body of the vial. Any angularity of the flange versus the vertical center line of the vial must be specified; so must the physical finish of the surface of the flange and internal neck bore to ensure satisfactory mating with the closure. Closures must be specified in terms of diameters, depth, thickness and elasticity.
This is the basis of a new article by Tim Sandle.

Tests to verify the container-closure can be grouped as microbial tests or non-microbial (physical) tests. Physical tests include the dye test; vacuum testing (typically leak testing with sensitivity to detect leaks down to approximately 5-10 microns); gas leakage determined using a bubble test; liquid leakage detected by atomic absorption of a copper ion tracer solution; laser-based gas headspace analysis using a frequency modulation spectroscopy; high voltage leak detection (which detects package defects using an electrical current); or a helium leak rate test (which quantitates the flow rate of helium from leaks in packaging after having been flooded with helium as a tracer gas).

Where microbial tests are required, there are various factors to consider. These form the basis of the paper.

Then reference is:

Sandle, T. (2017) Liquid Immersion Microbial Challenge Tests: Microbial Testing for Container Closure Integrity, Journal of Validation Technology, 23 (6): 1-10 (see:

Posted by Dr. Tim Sandle

Sunday, 11 March 2018

What to make of the draft EU GMP Annex 1?

In a forthright article for PharmTech, Russell Madsen, James Agalloco, James Akers take aim at the new draft Annex 1.

The authors state: “The revised Annex 1 on sterile manufacturing includes incorrect and ambiguous statements that must be fixed before implementation.”

The critique assesses:
  • Inconsistent use of and undefined terminology
  • Incorrect application of technology
  • Technical process errors
  • Application of risk analysis
  • Unusual concept of sterile product manufacture
The article can be read here: PharmTech

Posted by Dr. Tim Sandle

Saturday, 10 March 2018

Which microbes matter most?

Scientists have recently announced a major achievement in ecosystem science. Their research illustrates a powerful new technique to simultaneously measure the growth rates of hundreds of individual bacterial taxa in any given soil sample.

Until now, however, scientists have not had the ability to measure growth rates of individual microbial populations other than by studying them in pure cultures within a petri dish. Unlike larger organisms such as birds, fish or mammals that can be tagged and monitored over time, individual microbial species are difficult to study effectively in their natural environment.

The new measurement technique is based on the quantitative stable isotope probing (qSIP) technology. By adding rare stable isotopes to soil -- tracers that contain heavy oxygen (18O) -- and then sequencing the bacteria that incorporated that tracer, the scientists were able to measure microbial growth.

See: Northern Arizona University

Posted by Dr. Tim Sandle

Friday, 9 March 2018

Lab-on-a-chip for tracking single bacterial cells

Researchers at the Biozentrum of the University of Basel, together with researchers from the Max Planck Institute in Dresden, have set up a novel lab-on-a-chip with accompanying automatic analysis software.

Using the new system the researchers can now study precisely how genes are regulated in single cells under changing environmental conditions. This way, they do not only gain insights into gene regulatory processes but also an overview of the diversity of adaptive responses of bacteria to varying environments.

For example, it is possible to investigate how individual bacterial cells respond to a sudden exposure to an antibiotic: whether they die, stop growing, or simply continue to divide undisturbed. It is also possible to observe the antibiotic's increasing effect duration on the cells. This is important to understand why antibiotics do not always kill all pathogens.


Matthias Kaiser, Florian Jug, Thomas Julou, Siddharth Deshpande, Thomas Pfohl, Olin K. Silander, Gene Myers, Erik van Nimwegen. Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and softwareNature Communications, 2018; 9 (1) DOI: 10.1038/s41467-017-02505-0

Posted by Dr. Tim Sandle

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