By now you are probably aware of the importance of pre-treating stools samples to ensure maximum extraction efficiency. But did you know that inadequate vortexing of the pretreatment tubes can cause your parasitic samples to give false negative results?
Cryptosporidium and Giardia are parasites that contain a protective outer shell. This outer shell allows the organism to become resistant to environmental factors. Rigorous vortexing is critical in breaking open this outer shell to ensure efficient extraction.
We recommend vortexing the pretreatment tubes vigorously at a maximum speed for five minutes.
Mo Bio Laboratories, Inc. makes vortex adaptors (Cat. # 13000-V1-24) for your Vortex-Genie. What we love about this particular adaptor is that not only does it allow you to vortex up to 24 pretreatment tubes simultaneously, but it also orients the tubes in an angle that allows the beads to effectively break up any solid matter in the tube. This includes any parasites that may be in your sample.
Once the pretreated samples get aliquoted for extraction, the pretreatment tubes can be frozen at -80⁰C. To reuse the previously pre-treated tubes, simply thaw the tubes at room temperature. Once thawed, give each tube a quick 10 second vortex. Then centrifuge the tubes at 14,000 rpm for 2 minutes (to re-pellet the beads and any cellular debris). The samples are now ready to be extracted.
And that’s the scoop!
You’ve been using xTAG assays in your lab for a while, and now you’re ready to bring on xTAG Gastrointestinal Pathogen Panel (GPP) to detect viruses, bacteria, and parasites that cause gastroenteritis. If you want to properly differentiate and identify these pathogens, it is critical that you also differentiate GPP from other assays.
You already know the importance of pre-treating stool samples for GPP, and you’ve surely mastered the fine art of poop-scooping. You also know that you should never interchange reagents between different assay kits (for example, the reporter buffer and SAPE for GPP and RVP are different formulations). Today we’ll explore another major difference: GPP’s use of FAST chemistry and how it simplifies workflow and reduces turnaround time.
How Does it Work, and How Much Faster?
As a seasoned xTAG practitioner, you are aware of the traditional procedure: Extraction > Multiplex PCR > Amplicon Treatment > Allele-/Target-Specific Primer Extension > Bead Hybridization > Reporter Addition > Detection. Similarly, GPP utilizes a condensed version of this chemistry. Here’s an overview of the FAST workflow:
The first thing you’ll notice is the streamlined process, hence the FAST moniker. GPP can be performed in as little as five hours. This turnaround time is favorable when compared to other diagnostic methods such as stool culture or ova and parasite (O&P) exams.
As you dig deeper into the actual procedure, you may ask yourself, “Where’s the amplicon treatment and target-specific primer extension (TSPE)?” Before we get to the answer, let’s examine the purpose of each step.
Amplicon treatment is used to remove any unincorporated primers and dNTPs from the RT-PCR products. Removing these items is essential because the TSPE step will introduce new primers—with a TAG sequence incorporated at the 5’ end—and new dNTPS whose dCTPs are biotinylated. These TAGs and biotinylated-cytosines will allow us to later attach our beads and reporter molecules, respectively. Together, amplicon treatment and TSPE consume about two hours of thermal cycling time. To eliminate these separate reactions but maintain TSPE’s functionality, we have developed target-specific RT-PCR (illustrated below).
With this chemistry, the TAG sequences and biotin are now introduced during the initial RT-PCR step. Just like TSPE, a primer has a tag sequence on its 5’ end. But instead of using dCTPs, biotin is now incorporated on the 5’ end of the reverse primer. If the target is present, the double-stranded RT-PCR product will include a TAG on one end and biotin on the other. If the target is not present, the TAG and biotin will not be incorporated because extension will not occur.
These RT-PCR products are already equipped for bead hybridization. At this step there is another added bonus: the bead hybridization can be consolidated with reporter addition into one simple step. By the time this incubation is complete, the TAGs are coupled to their respective beads and the biotin has bonded with the reporter conjugate. You are now ready for data acquisition on a pre-heated analyzer. GPP is as simple and FAST as that!
Not So FAST, My Friend
Before you start thanking us for this enhanced chemistry (or questioning why we didn’t develop it sooner—or, dare I say, faster) we must discuss some precautions that must be taken when performing GPP. By consolidating many reactions with FAST chemistry, there are many molecular interactions occurring simultaneously. This can create opportunity for non-specific binding and the potential for false positive results. In contrast, it must also be noted that FAST chemistry reduces your chances of contamination by eliminating multiple manual handling steps. Nevertheless, these GPP precautions are universal for molecular assays but are of heightened significance with FAST chemistry.
1. Maintain unidirectional workflow
Designate rooms and/or workbenches for pre-PCR, template addition and amplification. Each area should have dedicated supplies and equipment.
2. Pay close attention to temperature
Reagents and samples should always be handled and/or stored at the appropriate temperatures and conditions specified by the package insert.
- Keep PCR reagents on a cold block when preparing master mix, especially the xTAG OneStep Enzyme.
- Keep prepared master mix and extracted samples cold during template addition.
- Pre-heat the thermal cycler to the specified temperatures for RT-PCR and bead hybridization steps.
Pre-heat the LX200 analyzer before data acquisition.
3. Work in an efficient, timely manner
Organize workbenches, supplies and techniques to minimize the time between hands-on steps.
- After extraction, transfer nucleic acid eluates to strip tubes or PCR plates instead of tubes that require individual manipulation.
- Use a repeater pipette to aliquot master mix and beads, especially for larger runs.
- Use multichannel pipettes for:
- Transferring extracted samples to master mix.
- Adding PCR products to beads.
- Adding and mixing reporter solution.
Let’s Quickly Wrap This Up
By employing xTAG GPP to detect gastroenteritis-causing pathogens, you can simplify labor for your lab and reduce turnaround time for your patients. Like all technological advancements, its FAST chemistry also poses its own set of challenges. However, if you follow the tips outlined above and do not deviate from the package insert, you can easily mitigate your chances for erroneous results. You should now be prepared to take on those potentially-infectious stool samples without trepidation.
Noroviruses are non-enveloped (naked) viruses with a positive, single-stranded RNA genome. They are the main cause of acute nonbacterial human gastroenteritis. Transmission of the virus occurs by food or by person to person via a fecal-oral route.
Noroviruses belong to the family Caliciviridae and are grouped into five genogroups ranging from GI through GV. These groups are further divided into at least 34 genotypes. GI and GII genogroups are the primary cause of human disease by noroviruses. Most outbreaks are caused by GII.4 strains.
Viruses with RNA as their genetic material can quickly adapt to changing environments and conditions. RNA viruses demonstrate genetic variation by mutation (due to the high error rates of viral polymerases that replicate their genomes), recombination, and reassortment. This ability to rapidly adapt to changing conditions leads to the emergence of new strains, often leading to an increase in outbreak activity. New GII.4 strains have been emerging every 2–3 years, replacing previously predominant GII.4 strains.
In March 2012, a new GII.4 norovirus strain, GII.4 Sydney, was identified in Australia and has since caused acute gastroenteritis outbreaks in multiple countries. During September–December 2012, a total of 141 (53%) of the 266 norovirus outbreaks were reported to CaliciNet—an electronic laboratory surveillance network maintained by the CDC—were caused by GII.4 Sydney. The other reported outbreaks were caused by 10 different GI and GII genotypes— including GII.4 New Orleans, which emerged in 2009.
The preferred method of detection of noroviruses is through molecular assays. Norovirus is detected by our FDA-approved xTAG® Gastrointestinal Pathogen Panel (GPP). This assay is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral, parasitic, and bacterial nucleic acids in human stool specimens from individuals showing signs and symptoms of infectious colitis or gastroenteritis.
Emerging noroviruses present an ongoing challenge to methods used to detect noroviruses. How do you know for sure that your norovirus assay will detect these emerging strains?
Norovirus primer sequences in our GPP panel are queried against the sequences of emerging strains, including GII.4 Sydney and GII.4 New Orleans, as they become available in the sequence databases. Our norovirus primers show 100% alignment to these 2 strains. A clinical specimen that was confirmed positive for GII.4 Sydney by a public health lab tested positive for Norovirus GII by the GPP panel.
Proactive monitoring of emerging strains will ensure consistent and reproducible detection of norovirus year after year by our GPP assay.
To learn more about noroviruses, see our Norovirus Infectious Information Sheet and the references below.
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CDC. Notes from the field: Emergence of new norovirus strain GII.4 Sydney—United States, 2012. Morbidity and Mortality Report Weekly Report. January 25, 2013. 62(03): 55-55.
Karst, S., M. Pathogenesis of noroviruses, emerging RNA virues. Viruses. 2010 March; 2(3): 748–781.
van Beek J, Ambert-Balay K, Botteldoorn N, Eden JS, Fonager J, Hewitt J, Iritani N, Kroneman A, Vennema H, Vinjé J, White PA, Koopmans M, on behalf of NoroNet. Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II.4, late 2012. Euro Surveill. 2013;18(1):pii=20345.
Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20345
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Also, see these recent Norovirus communications for more information:
Ricin is a proteinaceous toxin from the castor bean. When castor beans are processed to produce castor oil, the waste product, or “mash,” contains the ricin toxin. The toxin can then be purified from the mash. Eating castor beans can also be deadly as the ricin doesn’t have to be purified.
How much ricin does it take to make a person ill or kill them? An extremely small amount—roughly three milligrams is enough to be deadly, according to the US Centers for Disease Control. Ricin works by interfering with ribosomal function. Ribosomes are the subcellular components that make proteins in all types of cells. Since proteins are the functional and structural components of cells, cells start to lose their functionality and eventually die.
Accidental exposure to ricin is extremely unlikely: it generally has to be intentional, such as in the 2001 and 2013 episodes of ricin being sent through the U.S. Postal Service. If you are exposed to ricin, the symptoms occur within 6–12 hours. Typical symptoms include nausea, vomiting, and abdominal pain. Symptoms progress to include dehydration, internal organ failures. If inhaled, the toxin can cause respiratory symptoms first.
Numerous methods are currently used to identify ricin toxin. Usually an antigen-specific assay for screening samples is used, and then presumptive positive samples are re-screened with a confirmatory test. When multiple toxins are suspected, many initial screenings of the samples have to be performed.
Several research facilities have used Luminex Technology to identify ricin toxin in food and other samples. The U.S. FDA’s Center for Food Safety and Applied Nutrition, for instance, used xMAP® Technology to detect abrin, ricin, botulinim and Staphylococcus enterotoxins toxins in food. The Russian Academy of Sciences also used xMAP Technology to identify Staphylococcal enterotoxins, cholera toxin, ricin, botulinim toxin, and heat labile toxin of E. coli. Several government agencies of China utilized xMAP Technology to detect Bacillus anthracis (anthrax) spores, Yersinia pestis, SARS-CoV, Staphylococcal enterotoxin B (SEB) and ricin from powder samples.
For more information about xMAP Technology and how it can be used, please contact us.
Luminex’s IVD xTAG Gastrointestinal Pathogen Panel (GPP) utilizes a new version of our TDAS software, which employs many new user friendly features including the ability to choose which panels or specific targets to include or mask in the final report for each sample. In today’s “Tech Tuesday” we will walk through the GPP data analysis process and highlight these new features.
Once you have acquired your data, you are a few simple clicks away from viewing your data in the panel format of your choice. To analyze your data, click on the TDAS LSM icon on the desktop. The software will open and you will see the TDAS home screen. Choose “File” then “Open” (or simply click the file folder icon located at the upper left of the screen) and select the .csv file you wish to analyze.
Once the .csv file is selected, the software will prompt you to designate which samples in the run are negative controls (NC) and which (if any) have been diluted.
Each run must include at least one NC. Select “Negative Control” in the Sample type box and then click the sample ID for all samples that are negative controls. If any sample in the run has been re-run through the assay with 1/10 dilution of the extract, select Diluted Sample in the Sample type box, then click the Sample ID for those diluted samples. If there is no diluted sample in the run, select the check box “I confirm that NO diluted samples were run in this batch” and click “Next.”
The Mask Editor screen will appear next:
xTAG TDAS GPP US Version 1.2 allows the user the flexibility of choosing which panels or individual targets will be reported for each sample. If all viral, bacterial, or parasitic targets are desired, simply click the applicable word above the column and all samples will be checked for that panel (in our example here, all samples will include the viral panel, and some will include either or both of the Viral and Bacterial panels). If individual targets are to be chosen for each patient, the user can manually check or uncheck individual boxes as desired.
Note: Once analyzed, all subsequent TDAS analyses of this data will be performed using the same assay module and masking options for all samples. Click “Next” to proceed.
The user will then be asked to confirm their Mask Editor choices. All four boxes (Diluted Samples, Viral, Bacterial, and Parasitic) must be checked. Once analyzed, all subsequent TDAS analyses will be performed using the same module, the same set of diluted and negative control samples. You will not be able to unmask hidden targets or mask these selected targets. Click the “Back” button if modification is necessary. Click “Apply” to proceed.
The data is cleanly displayed in a summary view. For each sample, the calls for each of the targets tested will be listed from left to right.
Possible calls for targets include:
- POS: the target is detected.
- NEG: the target is not detected.
- No Call: there is a failure in one or more assay parameters / controls.
- – : call was masked by the user
Possible calls for controls include:
- PRES: the recommended Internal Control is detected.
- ABS: the recommended Internal Control is not detected.
- No Call: unable to determine presence or absence of the Internal Control due to an assay-specific criterion not being met.
The data can be displayed in a variety of additional report formats, allowing you to choose the format which best suits your laboratory’s needs. Clicking the “PDF” icon on the upper left of the tool bar will guide you through customizable PDF report creation. To view the data in Excel, click the “CSV” icon next to the “PDF” icon. If you prefer, you can simply print the summary screen straight from TDAS.
Many biological systems and populations are complex interactions of multiple components. These parts of cellular pathways or ecological systems often interact in ways with each other that require scientists to study as much of the system as possible, instead of trying to look at individual components. Below are four examples where studying multiple markers or species has led to greater understanding of systems.
Protein Biomarker Monitoring
Monitoring multiple proteins in saliva, plasma, and urine is desirable because these samples are easily obtained from patients. Evaluating patterns of expression of multiple biomarkers is becoming increasingly important to understand healthy versus diseased patients. Biological pathways are complex systems of interactions with many proteins involved in extracellular and intracellular signaling.
“Major advantages of multiplexing are: it decreases experimental variability, simultaneously detects numerous proteins from low sample volume, obtains quantitative results and is cost effective” (Khan 2012).
Specific disease processes, such as sepsis, involve several well-known protein markers. Sepsis is often a fatal disease caused by infection and involves varying numbers of inflammation biomarkers, depending upon the infectious agent causing the disease. Researchers at Copenhagen University used commercially available cytokine panels with added unique biomarkers in order to study the most relevant set of biomarkers, and did not rely upon studying single biomarkers at a time. Studying single markers one at a time often leads to missing important interactions of proteins. Researchers generally look for increases in proteins, but some proteins may increase in abundance and others may decrease during a disease process such as sepsis. Kofoed et al. expound on the advantages of multiplexing:
“Some of the advantages of multiplexing compared with measuring the same analytes by traditional ELISA are a reduction in pipetting error; a reduction in hands-on time and, therefore, cost; and improved quality of results because freezing/thawing would typically be required for the measurement of multiple analytes by ELISA. Another advantage is the reduced amount of sample needed, which is of particular importance in children, from whom small amounts of plasma are usually obtained, and in critically ill sepsis patients, for whom there is a need for monitoring immune status at several time points.”
In drug development, scientists need to understand how drug candidates are affecting the disease to be treated, as well as the health of the patient. Biomarkers are often used to perform these studies during development and in clinical trials. The major advantage of multiplexing is “that you gain a better understanding of the event you are measuring in the context of another parameter, minimizing faulty interpretation of data or ambiguity from data sets” (Tenorio-Borroto et al. 2012). Multiple biomarkers are used to monitor the effects of the drug and to insure that members of clinical trials don’t experience negative effects, such as a cytokine storm that happened during a clinical trial and led to severe issues for several members of the study. As stated by Marrer et al. (2007), “The advantages of multiplexing and microfluidics are manifold. First, the often limited sample volumes can be reduced especially when analyzing multiple biomarkers. Secondly, costs can be drastically reduced not only by analyzing several markers in a multiplex, but also by the reduction of sample volume and consequently the reduction of amount of reagents. Thirdly, multiplexing increases the throughput of measurements.”
Scientists take Luminex Technology and build assays for environmental monitoring to detect multiple organisms in samples. When samples are collected from the environment, scientists are often trying to detect multiple types of organisms such as bacteria, viruses, and parasites at the same time. Researchers need to monitor ocean water for the appearance of “red tides” caused by certain species of algae that are extremely toxic to other forms of marine life. Populations of aquatic animals can’t be identified in certain stages of their life cycle, which is why Gleason and Burton (2012) used multiplexing to identify fish egg species: “Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing.”
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1. Gleason, L. U.,Burton, R. S., (2012) High-Throughput Molecular Identification of Fish Eggs Using Multiplex Suspension Bead Arrays. Molecular Ecology Resources 12:57 – 66.
2. Khan, A., (2012) Detection and Quantitation of Forty Eight Cytokines, Chemokines, Growth Factors and Nine Acute Phase Proteins in Healthy Human Plasma, Saliva and Urine. Journal Of Proteomics 75:4802 – 4819.
3. Kofoed, K., Schneider, U., Scheel, T., Andersen, O.,Eugen-Olsen, J., (2006) Development and Validation of a Multiplex Add-on Assay for Sepsis Biomarkers Using Xmap Technology. Clinical Chemistry 52:1284-1293.
4. Marrer, E.,Dieterle, F., (2007) Promises of Biomarkers in Drug Development a Reality Check. Chemical Biology & Drug Design 69:381-394.
5. Tenorio-Borroto, E., Rivas, C. G. P., Chagoyan, J. C. V., Castanedo, N., Prado-Prado, F. J., Garcia-Mera, X.,Gonzalez-Diaz, H., (2012) Ann Multiplexing Model of Drugs Effect on Macrophages; Theoretical and Flow Cytometry Study on the Cytotoxicity of the Anti-Microbial Drug G1 in Spleen. Bioorganic & Medicinal Chemistry 20:6181 – 6194.
PCR has become a rapid and reliable tool for the molecular biology-based diagnosis of a variety of infectious diseases from a wide range of clinical samples. Fecal specimens, the sample type used in our GPP assay, are among the most complex specimens for PCR. Therefore, it must be emphasized that for such a sensitive and complex assay every element of PCR, no matter how simple or mundane, can affect the outcome of results. This increased sensitivity is critical for low copy pathogen detection and the amplification of degraded samples, as well as for multiplex PCR.
What happens prior to thermal cycling is critical to the success of PCR. We recommend paying particular attention to the temperature of the reagents during PCR set up because the Taq DNA polymerase retains some activity even at room temperature. Thus, under non-stringent annealing conditions, such as at room temperature, products can be generated from annealing of primers to target DNA at locations of low complementarity or having complementarity of just a few nucleotides at the 3′end. This can, in effect, create new templates attached with the primer sequences. Ensuing cycles amplify these attached sequences, generating nonspecific products and possibly reducing amplification efficiency of specific products by competition for substrates or polymerase.
Fortunately, focusing on keeping your reactions cold during PCR setup, preheating the thermocycler, and preheating the plate on the Luminex instrument will help you obtain consistently high-quality data and ensure the integrity and economic use of your GPP reagents. Remember, when it comes to PCR, shortcuts backfire more often than not.
The following problems can be a result of temperature-related issues:
- Consistently high background for a problematic target
- Random non-specific signal across several targets
- High background for most or all targets
- Low signal
To make your life easier, make sure to focus on the following points:
- Prior to beginning the assay, go through the GPP product insert and highlight the pre-heating reminders so you don’t overlook them.
- Keep everything cold! — Keep PCR reagents cold throughout Master Mix setup and Sample addition.
- Place PCR reagents in a cold block after thawing.
- Keep the Master Mix on ice or in a cold block.
- Use a PCR cooler rack (96-well cold block) to keep reaction aliquots cold during sample addition.
- Prepare Master Mix and add samples in a timely fashion, but don’t compromise proper technique or thorough mixing to save time.
- Pre-heat the thermal cycler to 53°C prior to RT-PCR.
- Quickly transfer the tubes from the cold block to the preheated thermocycler.
- Run in Block mode (BioRad and Eppendorf) with heated lid enabled.
- 53°C incubation temperature provides highly specific reverse transcription conditions resulting in lower background and increased yield of specific product.
- Pre-heat thermal cycler to 60 oC prior to Bead Hybridization to avoid high background.
- Perform Hybridization/Detection step within 12 hours of PCR completion.
- Pre-heat the Luminex XYP heater block to 45°C. Keeping the reactions at 45°C after the Bead Hybridization step until the time of data acquisition is essential for reduced background signals.
- The temperature of the Luminex XYP heater block should be set to 45°C at least 10 minutes before the completion of the hybridization/detection.
- The hybridization reaction should be transferred to the heated Luminex XYP block after the completion of the 45oC – 45 minute step. The additional 45oC – Hold step is to prevent the hybridization reaction from cooling if not immediately removed from the thermocycler.
- Efficiently transfer the hybridization reaction from the thermocycler to the Luminex to prevent cooling.
With the introduction of MagPlex® beads more streamlined protocols can be developed for protein and nucleic acid applications. With magnetic beads, high through put experiments being done in 96 well plates or higher formats no longer need to have washing and other buffer exchange steps done by spinning plates or washing beads with filter plates. Such steps increase processing times, prevent developing automated protocols, and contribute to bead loss and higher CVs. By switching MicroPlex® bead assays to MagPlex beads, protocol times can be shortened and lower bead loss generates more reliable and reproducible data.
Because a large number of different plate magnet designs are available, selection of the right plate magnet is critical to maximize the advantages of using MagPlex beads. Four different plate magnets designs include 1) flat plate magnets, 2) bar magnets, 3) post magnets, and 4) Ring magnets (Table 1). Several factors need to be considered to determine which magnet design fits your needs best. One of the first factors to consider is the type of assay it is used for, a protein based assay or nucleic acid assay with PCR steps and/or heated reading.
Many protein-based assays can be done in flat bottom microtiter plates and processed manually with or without plate washers. These types of assays can use flat plate magnets—where the entire bottom of the magnet is magnetized. While this type of magnet may be suitable for low sample number experiments processed manually, higher throughput experiments employing more automated handling may require a shift to bar-, post- or ring-type magnets—especially when they need to shift away from flat bottom plates and need plates with conical or curved wells. In this case, several other factors similar to those required for nucleic acid assays need to be considered as described below.
Most nucleic acid assays—especially those with PCR steps—can be processed manually or with semi-automated protocols. Due to the sensitivity of the PCR steps, it is crucial to prevent cross contamination between wells on the plate. As a result, whether using a manual processing protocol or automated protocol, disposable tips are required to eliminate any cross contamination between wells on the plates. In addition, the need to run reactions in a thermal cycler or analyze in the Luminex heater block requires the use of PCR plates with conical shaped wells.
During different steps in the protocols and during washing steps, these assay characteristics require the complete removal of buffers from the wells with minimal bead loss. This requires a magnet design that will pull the beads to the sides of the wells so that pipette tips can go all the way to the bottom for complete removal of reaction buffers. For PCR-based nucleic acid assays and high through put protein assays employing conical well plates, the two following factors must to be considered.
- At any step in the protocol when the magnet is needed and the plate fits securely on the magnet, is the reaction volume large enough that the top of the solution is above the top of the magnet? If the top of the solution is below the top of the magnet, beads can be pulled out of the solution dramatically altering the reliability of the assay (Figure 1).
- Is the magnet strong enough to pull the beads out of the way in a short period of time? Reaction solutions can have different viscosities and will require different strength magnets. So the strongest magnet that also meets the needs of requirement 1 above will be the preferred magnet—especially if it can clear the solution of beads within 2 minutes or less.
So whether you are designing a multiplex proteomic assay or nucleic acid assay, keep these guidelines in mind when selecting a plate magnet. If you need further recommendations for the best magnet for your specific Luminex application, contact Luminex technical support or your Field Application Scientist for more information.
“Humans can get salmonella from chickens by touching them or their manure, according to the CDC. The birds can spread the bacteria even when they look healthy. The agency says the best way to reduce risk is to wash hands after handling birds — and make sure that children wash their hands, too.”
“The parasite that caused the [Cryptosporidium] outbreak is thought to have originated in ready-to-eat bagged salad mixes, said England’s Health Protection Agency in a report published Tuesday.”
“Parasites of various kinds rely on being swallowed in order to infect a human host. Three protozoa in particular, however, are responsible for the majority of documented outbreaks of foodborne parasitic illness: Cryptosporidium spp., Giardia duodenalis (also known as Giardia lamblia) and Cyclospora cayetanensis. Such outbreaks are less common than those caused by bacteria, and it’s tempting to think that the presence of protozoa on our produce is a rare exception, but recent evidence suggests that it may be far more common than we think.”
“On the basis of their new analysis, Dr. Payne and colleagues estimate the average annual numbers of US hospitalizations, emergency department visits, and outpatient visits due to norovirus infection for kids under 5 exceeded 14,000, 281,000, and 627,000, respectively, in 2009 and 2010. The estimated cost for care was more than $273 million in treatment costs each year.”
“Then an epiphany. Burke turned 30. He read that life expectancy was 28. Small but significant advances were tacking on precious time for people with cystic fibrosis, maybe one advance every five or so years.”
“P. aeruginosa infections easily develop in the excess mucus accumulating in the lungs of patients with CF, according to the FDA. One antibiotic remedy in the past has been tobramycin inhalation solution (TOBI, Novartis), on the market since 1997. It is delivered through a nebulizer. In contrast, tobramycin inhalation powder (TIP) is delivered through a hand-held, pocket-sized inhaler.”
“The study, published in the journal Food and Environmental Virology, found when using uncontaminated utensils, more than half of all knives and graters were contaminated after preparing the contaminated produce.”
“The “superbug,” E. coli ST131, was first discovered in 2008 and is immune to most standard antibiotics. Though infections were rare in the first few years after the strain was discovered, it accounted for more than a quarter of the 299 infections that researchers at the University of Minnesota studied from Olmsted County in Minnesota between February and March of 2011.”
“’We had been feeling sick for a few days in a hotel room, thought we had the flu and decided — maybe we didn’t,” Marker says during a visit to StoryCorps in Santa Fe. “And, of course, it was a good thing we got to the hospital because it was the first case of plague in New York City in over 100 years.’”
“The “sonopill” would relay ultrasound images from inside the body after being swallowed by patients. It is hoped it could allow gastrointestinal problems to be diagnosed without the need for uncomfortable endoscopic exams.”
“While the Mediterranean liquid gold has long been touted for its multiple health benefits, a team of scientists from the University of Louisiana identified a specific component in olive oil that they say protects nerve cells from damage brought on by Alzheimer’s disease.”
“The weekly flu report of the Centers for Disease Control and Prevention in Atlanta said for the week ending March 17, influenza activity remained elevated in the United States, but instead of the influenza A viruses that dominated during most of the flu season, influenza B viruses were predominant and 72 percent of all viruses reported were influenza B.”
“Tap water in Shanghai is taken from near the center of the river, at the bottom, where the water quality is better, Shentu says. And local water authorities have upped the chlorine in Shanghai’s water, to improve sterilization.”
“Caffeine and methylxanthine come to us by way of chocolate, sodas, energy drinks, tea and coffee — and many of us are undeniably grateful for that. But as a result of their popularity, these compounds have become a common pollutant in wastewater and surface waters. Caffeine is toxic to a variety of organisms and can wreak havoc on ecosystems; it alters natural bacterial flora and inhibits the germination and growth of some plants.”
“Overall, 195 people across 27 states were infected during the outbreak, which involved three salmonella strains. Eighty-seven percent of infected people for whom purchasing information was available bought chicks or ducklings from a single mail-order hatchery in Ohio, which has been linked to other salmonella outbreaks.”
“In the past, searches in northern Utah have required dozens of officers and the use of ATVs, snowmobiles and other equipment. Fielding said the bracelets also could be used for autistic children or anyone at risk of wandering off, adding that police would monitor the person’s movement only when the family contacted them.”
Why do researchers want to measure multiple intercellular proteins? Well, many are potential biomarkers and by measuring multiple proteins, researchers can get a clearer picture of what is happening in biological pathways.
This publication is significant because Dr. Alamgir Khan measured three different sample types, urine, saliva and plasma, to get a better understanding of expression levels in different bodily fluids. The measurements were all performed using Bio-Plex® multiplex kits, which are based on Luminex Technology. In prior work, which Dr. Khan summarizes in the paper, different methods were used at different times and with different sample types. The new publication shows that most of the biomarkers studied are detectable in healthy men and women and in all three sample types, providing a basis for more researchers to have confidence that they can simultaneously measure the majority of these biomarkers simultaneously and in the three sample types. This is important in biomarker profiling of different disease states where cytokines, chemokines, and growth factors play a role.
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Australian Proteome Analysis Facility, Macquarie University, Sydney NSW 2109, Australia. [email protected]
Cytokines, chemokines, growth factors (CCGFs) and other low abundance proteins/peptides in human body fluids or in tissues are potential biomarkers. Human body fluids such as plasma, saliva, urine, etc. are being analyzed more frequently than tissues primarily because of ease of sample collection. However, available information on concentrations of a large number of CCGFs in various body fluids of the same healthy individuals and gender-specific CCGFs is limited. In this work concentrations of 48 CCGFs were measured using multiplex bead assays and compared between plasma, saliva and urine collected from 20 male and female healthy volunteers. Forty three CCGFs were detected at least in one sample type of which 37 were in plasma, 41 were in saliva, and 34 were in urine; five CCGFs were not detected in any sample. Concentrations of detected CCGFs differed significantly between sample types but similar between gender groups. Gender-specific CCGFs were also observed. Concentrations of nine acute phase proteins were also measured from plasma, saliva and urine to determine general health conditions of the volunteers. This work will provide an idea of which CCGFs are detectable and their relative concentrations in healthy human plasma, saliva and urine and which CCGFs are gender-specific.