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8. This story
If you read only one story today about a college hockey player having his season ruined by a 25-inch intestinal parasite, make it this deeply unpleasant one.
7. Losing Mark Hunter
This was the kind of change that you knew was going to happen, right?
You have to say, well the Leafs gave the right guy the GM job, so there was no way Hunter was going to stick around and let Dubas be his boss, especially not when he has other options available perhaps including for other teams' GM jobs if, say, things open up.
Interesting, though, that he can't take a new job elsewhere until July. Wonder what that means for what the Leafs are planning at the draft, in free agency, etc., and how Hunter could potentially throw a wrench in that. Obviously you don't want a guy who just sat in on months' worth of meetings talking to a new team about everything you want to do, but Lou Lamoriello was under no such restrictions, so y'know.
Speaking of which…
6. Lou on the Island
"Full authority." This should be a worrisome phrase for Islanders fans. There is certainly the plausible deniability among the analytics crowd that comes with being able to say "Every bad move the Leafs made was Lou's fault, and every good one was Dubas's idea" but the most likely scenario is that a bunch of guys sat around in a meeting and talked out trades and signings until a consensus could be formed.
This is, from what I understand, how lots of organizations work and that's good. You want groups of smart people making decisions instead of things being done unilaterally by one guy.
And maybe that's what will happen in Brooklyn too, right? But full authority for Lou Lamoriello might mean a lot more of "signing guys like Matt Martin" than "signing guys like John Tavares." Which, I guess that's not entirely fair because the Tavares thing has nothing to do with Lamoriello being there or not being there, but you see the point.
The kind of hockey Lamoriello seems to think works in 2018 is more Casey Cizikas than Mat Barzal.
5. David Quinn
Really interesting "pending" hire by the Rangers.
On the one hand I never thought Quinn would leave Boston University, his alma mater where he earned a very good salary and was widely hailed as one of the best coaches in college hockey. Sure, you want to test yourself professionally, but you probably need one hell of an offer to leave a cushy gig like that.
The Rangers gave it to him. He will make in five years something akin to what he would have earned in 10 to 20 years at BU (depending upon whose reports you believe about his BU salary). That's a life-changing offer, and not least because even guys who flame out hard as NHL head coaches will always be able to get a job in pro hockey, somewhere.
The question, I guess, is what the Rangers expect from Quinn. Anyone who's around college hockey a decent amount knows Quinn is regarded as a great communicator and motivator, especially for younger high-end guys. That's something the Rangers definitely need as they start their rebuild in earnest. His teams also play a modern, up-tempo brand of hockey. However, it's also fair to say that Quinn's results, routinely with one of the two or three most-talented teams in the NCAA, have been a little lacking. One Beanpot, two regular-season league titles, two postseason titles, one NCAA final appearance (with the worst third-period meltdown in recent memory). A lot of that came because Jack Eichel came to campus, which was of course Quinn's doing.
So if you're concerned about the Rangers underperforming with Quinn, well the good news is they're rebuilding and as long as young players get better, he's doing his job. Wins and losses shouldn't really enter into the discussion too much if other indicators are there.
But at the same time, well, this is a lot of money for a totally unproven coach who, by the way, was seemingly their second choice. Jim Montgomery, now the coach of the Dallas Stars and ex of Denver University, reportedly turned the Rangers down first. Interesting times.
4. Managing the Jets' cap
Everyone seems to think the Jets will be very good for a very long time but I went to their CapFriendly page after they lost the conference final and jeeeeez.
They have to re-sign nine restricted free agents this summer, including useful players like Brandon Tanev and Joel Armia (who won't be that expensive), Josh Morrissey (maybe a little expensive?), and both Jacob Trouba and Connor Hellebuyck (very expensive).
Wonder where that leaves them, right? Because it would be nice to bring back Toby Enstrom and Paul Stastny, but how much cap space will be available for them, especially since they will also have to re-sign Kyle Connor and Patrik Laine in summer 2019 as well. Yeah the cap is going to go up for next season, so Winnipeg probably has like $25 million to spend on all the guys they need to re-sign, but you gotta keep some of that powder dry because Laine and Connor are going to cost a fortune.
It's nice to have all these players and be able to meaningfully compete for the next few years, but I can't envy Kevin Cheveldayoff putting together a five-year plan for this stuff.
That being said, it's a nice problem to have.
3. Paul Fenton
A less-nice problem to have: Being the guy whose job is now to wring more out of this Wild group.
Paul Fenton has the ignominious task of improving this group that's already largely locked in for another year or two and doesn't really have a lot high-end talent coming any time soon. They have about $12-13 million to spend this summer, with only four non-star RFAs to re-sign (Jason Zucker and Matt Dumba certainly top-line the group).
Most of the roles are spoken for. There's not much in the way of roster space for a, say, second-line scoring winger available. So I guess Fenton's job is to improve the prospect pool or something, right? He should do well with that. That's his whole thing.
But as with Quinn, you wonder what the expectations are from his bosses. Should this team be able to meaningfully compete with Winnipeg and Nashville, the two best teams in the league that also happen to be in their division, under the current playoff format? I don't think that's reasonable to put on anyone.
2. Not Alex Ovechkin
That's a big Game 6, baby.
Ovechkin didn't score or even have an assist — Devante Smith-Pelly did, though, baby!!!! — but he was active. Seven shot attempts, four on net, in less than 17 minutes (what the hell?).
Instead, it was Nicky Backstrom who brought the serious heat: the Caps out-attempted Tampa 23-8 with him on the ice and he had two assists, including an absolutely clinical one to T.J. Oshie to open the scoring.
Ovechkin, on the other hand, was content to just try to murder people and unload it on the power play. Which I guess is his whole thing.
1. Game 7
We haven't had enough of these and frankly it's good that at least there is some drama for either the Bolts or a conference final on the whole. Let's goooooo!
(Not ranked this week: "Don't call them a Cinderella team."
Dawg if you don't think an expansion team making it to the Cup Final in Year 1 is some serious fairy tale material, might I remind you that they have a guy in a suit of armor come out and fight mythical beasts and stuff before every game.)
Ryan Lambert is a Puck Daddy columnist. His email is here and his Twitter is here.
(All statistics via Corsica unless otherwise noted.)


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The Best Things About International Disney Parks
10 Reasons You Need to Add an International Disney Park to Your 2019 Bucket List
10 Reasons You Need to Add an International Disney Park to Your 2019 Bucket List
Did you know Disney has 12 theme parks throughout the world? With so much magic to experience, 2019 is definitely the year to spread your Tinker Bell wings and visit one of their international parks. With options in France, Japan, and China, there's quite a few places to get both your Mickey fix and a completely different cultural experience for your family. Whether you want to feel like you're right in the middle of Ratatouille in Disneyland Paris or be transported to a whole new world at Tokyo Disney Sea, there really is something for everyone. So pack up the kiddos, keep reading, and we'll see you around the world!

subset: commoncrawl


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In This Section
Illegal dumping is the deliberate disposal of materials onto public land without a licence, permit or approval from the relevant authority. It is a criminal offence, with a maximum penalty of $5,000.
Illegal dumping is a concern as it can cause environmental harm and can be a public safety risk, especially when there are sharp objects and asbestos involved.
Illegally dumped waste is also expensive for Council and the community to clean up.
If you have any items to dispose of, please see information on our website under the following categories:
If you see illegal dumping activity on public land please contact the Ranger Services at the City of Burnside on 8366 4200.
To report an abandoned shopping trolley in your street, please follow the details below to have it removed:
Phone: 1800 876 553
Report an abandoned trolley
Phone: 1800 641 497
If the trolley belongs to a business not listed above, please contact the business directly to notify them of the trolley and location.
Civic Centre
401 Greenhill Rd,
Tusmore SA 5065
Mon - Fri 8.30 am to 5 pm
Public Holidays CLOSED


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Remember to Verify Patients' Eligibility and Benefits in the New Year
With the new year soon upon us, doctors are urged to be diligent in verifying patients' eligibility and benefits to help ensure they will be paid for services rendered. The beginning of a new year also means that both calendar year deductibles and visit frequency limitations reset. And, with the January 31 end of California's open enrollment period, patients may even be covered by a new payor.
The new year also brings a host of other challenges that could affect your ability to be paid:
- On January 1, 2018, Health Net Federal Services (HNFS) began providing managed care services to 2.9 million TRICARE beneficiaries in the 21 western states, including California. HNFS will be taking over the contract previously held by United Healthcare Military and Veterans' Services.
- The Covered California open enrollment period began November 1 and ended January 31, 2018. While Covered California will maintain its relationship with all 11 health plans that participated in the California exchange in 2017, Anthem Blue Cross is exiting the exchange market in all but three regions in 2018, citing market instability. This will impact over 150,000 enrollees.
Don't get stuck with unnecessary denials or an upset patient. Before the patient arrives obtain updated insurance information and verify eligibility at the time of scheduling, if possible, and making copies of the insurance card at the time of the visit.
And, don't forget that deductibles are typically based on the calendar year and reset on January 1. Best practice is to communicate with patients upon scheduling to remind them that their plan has a deductible that may have reset on January 1 and, if that is the case, that payment will be due at the time of service. If you offer an appointment reminder service, remind the patient if payment is expected at the time of service. Failure to collect deductibles, copays and coinsurance at the time of service can be very costly for a practice, as your ability to collect can decrease significantly after the patient leaves the office.
Taking these proactive steps to protect your practice by preventing denials, delays in payment and disgruntled patients goes a long way toward ultimately saving time and money.


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InstantMoneyClub.com is an online membership organization, designed to generate instant money for its members and whose goal is "to send money to every one of its members. Lots of money. We set no limit as to how much money a member can receive."
InstantMoneyClub.com was founded by a former Wall Street professional who has managed money market portfolios of up to half-a-billion dollars, understands money, and now spends his time helping people get more of it.
Adult content and gaming sites are eligible for the free memberships.
For free membership click on "webmasters" at InstantMoneyClub.com


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Oprah Winfrey has a sister
Like many things on Oprah's show, this sounds like a soap opera plot, but it's actually the real deal – Oprah Winfrey recently discovered that she has a half-sister she never knew about, and they had a big, tearful reunion on the show this week.
Her sister's name is Patricia, she's nine years younger than Oprah, and according to Oprah, she never even knew her mother was pregnant. Â It's difficult enough to imagine what an emotional thing it must be to find your family, no matter who they end up being, but to discover that your sister is Oprah? Â That's just crazy. Â It's like winning the lottery twice in a row!
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In my previous post I briefly mentioned Database Appliance with Project Indiana. Now that Sun has acquired MySQL (or as some people say MySQL has acquired Sun) and with our existing work in progress with PostgreSQL, there are quite a bit of options available of using some combination of Storage, System, Operating System, Database along with some end user application and present it as either Database Appliance or use it as Embedded Database.
I thought I will just discuss the audience, symptoms, merits, cons etc regarding the two approach and how they can be useful.
The first question is who likes database appliances and who wants embedded database? To answer that I would put the question back: What do you in hand? A hammer or a screw-driver? Because if you have a hammer, the whole world is a nail to you and if you have a screw driver the whole world is full of screws. Similarly if you love databases (DBA) and you want to query everything via a SQL statement then database appliances are for you. If you dont like to interact with databases (System Administrator, Application User) then you essentially want an embedded database where the OS or your end application takes care of interacting with the database. Well that is quite simple.
First lets talk about database appliance. Think of it as your existing wireless routers. You bring it in house, plug it into your network (essentially to your cable modem) and power it on. Connect your laptop to one of the LAN ports, run DHCP, fire up a browser and go to typically htp://192.168.1.1 and lo and behold you get a website to login and administer your router. A typical database appliance in some way will behave similarly. It has a web-based administrator to set it up quickly and allow you to connect to the database via rest of your applications on the network. In fact all the pieces are already out there: Storage, Server, Operating System, Database and even a web based management tool like webconsole (to configure ZFS) and Webmin which even supports mysql. This help reduce the knowledge of underpinning Operating System that you trust to "just work". Once setup, your applications just connect through your applications talking to client/server setup of a normal database. While the appliance takes care of snapshots, backups, clones via the web-based tool. Can a database be easier than that? That's the values that a database appliance brings to the table. Its DBA's dream to not go through a System Administrator to allocate the OS resources for the database. It abstracts the resilient operating system underneath it and makes it easier for the DBA to quickly deploy it in actual usage.
What about Embedded Databases? In some ways it is anti-database appliance. Other way to look at it is abstraction of the database functions itself via end application Functions which interacts with the database. The application can be either Operating System or the end application. One of the analogy that can be used here is an integrated database in the operating system which is controlled via services of the operating system. For example if you use "svcadm enable postgres:version_82" on Solaris, a PostgreSQL 8.2 instance is enabled for you in the background. Which means you are ready to use it with no idea on where it is or what it is (except from the svcadm name). That's an analogy of embedded database. Many applications infact create their own menu items to backup application which includes the database underneath it and so on. If things are setup right, then you dont even have to execute any dedicated PostgreSQL commands to stop/start, backup etc and everything is done using application like svcadm, zfs snapshot, etc.
After all it is all about choice and what one desires. Options and components already available. As database becomes a commodity, I wouldnt be surprised to see small consumer devices with a hard disk presenting itself as a MySQL or PostgreSQL database for structured information and as a NAS device for unstructured information. Talk about being a commodity.


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INSPIRE BOXES are supported by the Fetzer Institute as a means of highlighting exemplary initiatives or people promoting peace and reconciliation
In 2002, AKDN began to rehabilitate the Bagh-e-Babur (Babur's Gardens), an early 16th century terraced domain that includes the tomb of Zahiruddin Muhammad Babur, the founder of the Moghal Empire. AKDN's restoration, which has been underway for six years, began with the reconstruction of large sections of the 1.5 kilometre long Pakhsa (baked mud) perimeter walls. This phase of the reconstruction culminated with the rehabilitation of the Queen's Palace, a late 19th century structure built by Amir Abdurrahman Khan. In the process, rehabilitation teams excavated and reconstructed the historic water channels and tanks, which provided irrigation for the garden until the early 20th century. They also restored Babur's tomb along with the 17th century Shahjahan Mosque, which now conform to their original designs. At the foot of the garden near the Kabul River, a reconstructed caravanserai, similar to those found in Baku and elsewhere in Central Asia, now provides space for a visitor's centre, shops and offices.
Wherever possible the garden's rich horticulture, including fruit trees and plants, which were described in meticulous detail by Babur himself in the Baburnama (Babur's memoirs), has been restored. A crucial part of the rehabilitation work involved improving access, drainage and flood protection facilities for the 10,000 inhabitants of the surrounding residential area. Rare bird and other wildlife have now begun to return.
Since early 2008, Babur's Gardens have been maintained and operated by an independent trust, which managed to become self-sustaining in 2010, enabling hundreds of thousands of visitors to use the site for cultural and recreational purposes each year.
In a similar project, Jolyon Leslie, a South African architect and one of the country's most experienced cultural experts, has been working on the renovation of the war-damaged quarters of the Old City of Kabul, once a crossroads of civilizations. An elaborate blend of Indian, Persian and Central Asian influences is noticeable, not only in the Old City's unique architecture, but also in its arts and crafts. War shattered much of the Old City's craft industry, leaving most of its inhabitants impoverished.
The AKDN has sought to conserve key historic buildings, including houses, mosques, shrines and public facilities, and the transformation since the programme's launch has been stunning. Upgrading works have dramatically improved living conditions for some 35,000 Old City residents. Socio-economic training for hundreds of households in the neighbourhoods of Asheqan wa Arefan, Chindawol and Kuche Kharabat have fostered newly found skills in tailoring, embroidery, and weaving. The recent reclamation of Baghe Qazi (Judge's Garden), a 3.5-hectare public garden, located in the heart of the Old City, will also enable local residents to benefit from using the green space for recreational and sporting activities.
The Turquoise Foundation, which started its Old City rehabilitation work in 2006, has also contributed to the restoration. Like the AKDN, the Turquoise Foundation has rebuilt homes, improved basic infrastructure and preserved historic buildings. It has also focused on rebuilding the country's neglected craftwork by establishing the Institute for Afghan Arts and Architecture, which offers schools in woodworking, calligraphy & painting, ceramics and jewellery & gem-cutting. It has also opened a school in Murad Khane, the artistic quarter of Kabul's Old City. It ahs helped sell nearly one million US dollars worth of traditional Afghan arts and crafts both at home and abroad.
Although the work so far has been impressive, more remains to be accomplished. Many other historic buildings in the capital and in other Afghan cities have been neglected for too long and are now in danger of collapsing. Some of these buildings have also suffered from deliberate destruction by ruthless real estate entrepreneurs and corrupt government officials. Some buildings are knocked down overnight in a bid to avoid protection orders, which, in any case, are frequently not enforced if the police have been paid off.
In 2009 the AKDN completed the restoration of the imposing 19th-century brick mausoleum of Timur Shah in central Kabul. Responsible for consolidating the political gains made by his father Ahmad Shah Durrani, considered by many as the founder of the modern state of Afghanistan, Timur Shah was responsible for transferring the seat of the power from Kandahar to Kabul. The remaining open space surrounding the mausoleum has been reclaimed to serve as a public garden in a densely crowded part of the city.
Renovation in other cities
But Kabul is not the only place where urgently needed renovation is being carried out. In the ancient western Afghan city of Herat, well known for its historic architecture, the AKDN has worked at upgrading and documenting surviving historic quarters and monuments of the Old City. This includes the restoration of the Ikhtyaruddin Citadel and the Char Suq cistern. It has undertaken additional rehabilitation work in the Timurid era shrine complex built for Abdullah Ansari in Gazurgah, northeast of the city.
As part of a further initiative aimed at preserving and developing Afghanistan's cultural heritage, AKDN has established schools of classical Afghan music in Kabul and Herat. Years of war had threatened the survival of the country's classical music tradition. Under the Ustad-Shagird training scheme begun in 2003, master musicians teach small groups of students, selected on merit, ensuring the transfer their musical skills to a younger generation of performers. Instruments taught include the rubab, delruba, sarinda, dutar and sitar.
The restoration of urban monuments, houses and public spaces, as well as related cultural programmes, have been important sources of training for craftsmen, creating employment and facilitating economic regeneration. But perhaps most importantly, the work undertaken by AKDN, Turquoise Foundation and other organizations is also creating a sense of normality and hope in a country deeply affected not only by decades of conflict, but also religious and ethnic division. Albeit costly, the investment in cultural heritage needs to become of part basic development if Afghanistan is ever to emerge from its current quagmire.
Contacts: Aga Khan Foundation Afghanistan.


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Yaochuang Energy professional 1KW - 10KW solar panels for home irrigation water pump & lighting
Why choose cooperate with YAOCHUANG ENERGY?1.YAOCHUANG ENERGY works on solar power system over 10 years begin with some engineers.2.Important equipments of system are solar panel & solar pump inverter & water pump, solar panel & pump inverter are manufactured by ourselves, water pumps are from cooperated factory, and some famous brand pumps.3.Rich experience on design, and installation, full service. We are not only solar energy products producer, but also solar power system integrator & installer that cooperate with government.4.Hundreds projects in Africa, southeast Asia, and the Middle East, praised quality & service, positive feedback.5.Whole set system 1 year warranty, solar panel 10 years warranty, solar pump inverter & water pump 1 year warranty, lead acid solar battery 2 years warranty.
6.Commissioning and installation service is available all over the world.
Submersible deep well water pump is the most common used type for agriculture irrigation
How to establish business with us?1.Contacting us via email or trademanager, tell us what products or system you need, and your requirements2.Specification & price will be sent to you within 12 hours after getting your inquiry (except for holidays)3.Adjusting design and price according to your budget until you accept it4.Preparing products within 5 days once deposit is arrived, and ship out parcel after receiving balance payment (please give us some more prepared time for bulk order)5.Informing you freight information, and offering you bill of loading6.Guiding installating after you receive products7.Giving us feedback, and repeat order
You are welcomed to contact us for more details of hybrid solar power system.


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U.S. Equal Employment
Opportunity Commission
WASHINGTON -- The U.S. Equal Employment Opportunity Commission (EEOC) will hold a meeting on Wednesday, April 5, from 9:30 a.m. to 12:30 p.m. (Eastern Time), at agency headquarters, 131 M Street, N.E., Washington D.C. The meeting, entitled "The State of the Workforce and the Future of Work," will be open to public observation.
The meeting will consist of a discussion with the following panelists who are confirmed to participate:
Seating is limited. We encourage visitors to arrive 30 minutes before the meeting to be processed through security and escorted to the meeting room. Visitors should bring a government-issued photo identification card to facilitate entry into the building.
The EEOC advances opportunity in the workplace by enforcing federal laws prohibiting employment discrimination. More information is available at www.eeoc.gov. Stay connected with the latest EEOC news by subscribing to our email updates.


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Passion, Imagination, Care and Trust – these are the values that make Spintelligent different.
Passion and imagination drive creative solutions and new ideas to deliver results for our customers. Care for and trust in our customers and partners underpin how we work and the service we deliver.
We are continually on the look-out for like-minded people who share our passion in who we are and what we do. As an employer, we support employees in finding a work/life balance and we recognise the importance of a vibrant and dynamic working environment.
When you join Spintelligent you become part of a company that is passionate about growing and nurturing talent. Development opportunities come in various shapes and sizes dependant on your job, your experience and your future plans. We are proud to give our employees the tools they need to continue to grow and succeed.
Exciting. Challenging. Rewarding.
The range of careers available at Spintelligent is enormous. As part of Clarion Group, we operate from 9 offices in the, UK, USA, UAE, Brazil, Germany, Singapore, South Africa, The Netherlands and Turkey. We're constantly looking for people with the drive and talent to fulfil a broad range of roles across all our functions, and who can also demonstrate our core values. We are currently looking for experienced sales and marketing professionals, if you match this description we would love to hear from you.
Contact Louise Krull for further information:
Email: email@example.com | Phone: +27 21 700 3500


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This is the McAllister Racing '57 Ford Bomber body. Originally designed for on-road cars, the details on this makes it a great candidate for your Avenger monster truck replicas. There are no Ford logos or emblems molded into the body, and we feel this makes a better base for a replica than the '55 Chevy version.
FEATURES: Made of durable lexan plastic
Perfect fit and porportions for the Clodbuster
INCLUDES: One unpainted '57 Ford Bomber body
One window mask sheet
SPECS: Length: 18" (457mm) Width: 7.75" (197mm) Height: 4.6" (115mm) Wheelbase: 10.5" (267mm)
Shipping Charges: Our standard shipping charge for this product is $6.50 for the 1st item and $2.00 for each additional body.


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I have a Christmas card to share today using one of the older images from DigiStamp Boutique. This is just one from the set ....
Don't forget you can enter any of your creations using a Digi Stamp Boutique image into the monthly competition. The prize is a fabulous £15 voucher for the Digi Stamp Boutique Website.
I'd like to enter the following challenges :-
Crafty Hazelnuts Extra - Anything Christmas
Thanks for taking the time to visit my little blog today and for all your comments and encouragement.


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World Security Report2017-06-21 08:06:00
OnSSI has announced the release of Ocularis version 5.4, the latest version to the company's industry leading Video Management Software (VMS) solution.
This newest release further expands Ocularis' ability to provide end users with a comprehensive yet easy-to-use video management platform that accommodates the latest industry trends and expands on-the-go coverage.
New to Ocularis 5.4 is support for H.265 compression for exceptional image quality with less data, and a reduction of video bandwidth usage and storage requirements. Additional features include installation improvements including increased SQL server flexibility; new view layouts in Ocularis Client; and expanded Ocularis 5 Web and Mobile features including M2O™ (Mobile-to-Ocularis) technology, alert handling, triggers, AVI exporting and Ocularis toast notifications.
"Ocularis continues to evolve with enhancements that fulfill users' requests and address the latest technology trends impacting our industry," said Ken LaMarca, VP of Sales & Marketing, OnSSI. "The bountiful enhancements of Ocularis 5.4 will help users achieve more customized and better results via the improved image quality, on-the-go video capture capability and operational refinements in this latest release."
For more information contact:


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- Research article
- Open Access
Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess
- Subhash C Parija†1Email author and
- Krishna Khairnar†1
© Parija and Khairnar; licensee BioMed Central Ltd. 2007
- Received: 07 December 2006
- Accepted: 18 May 2007
- Published: 18 May 2007
Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA.
E. histolytica DNA was detected in liver abscess pus specimen of 80.4% of ALA patients by a nested multiplex polymerase chain reaction (PCR) targeting 16S-like r RNA gene. The nested PCR detected E. histolytica DNA in all 37 (100%) liver abscess pus specimens collected prior to metronidazole treatment, but were detected in only 53 of 75 (70.6%) pus specimens collected after therapy with metronidazole. Similarly, the PCR detected E. histolytica DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected E. histolytica DNA in only 4 of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but were detected in 17 of 30 (56.7%) urine specimens collected after treatment with metronidazole. The enzyme-linked immunosorbent assay (ELISA) for the detection of lectin E. histolytica antigen in the liver abscess pus showed a sensitivity of 50% and the indirect haemagglutination (IHA) test for detection of amoebic antibodies in the serum showed a sensitivity of 76.8% for the diagnosis of the ALA.
The present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica DNA in urine of patients with ALA can also be used as a prognostic marker to assess the course of the diseases following therapy by metronidazole. The detection of E. histolytica DNA in urine specimen of ALA patients provides a new approach for the diagnosis of ALA.
- Metronidazole
- Liver Abscess
- Urine Specimen
- Cystic Echinococcosis
- Pyogenic Liver Abscess
Infection with Entamoeba histolytica, results in 34 million to 50 million symptomatic cases of amoebiasis worldwide each year, causing 40 to 100 thousand deaths annually [1]. Mortality from amoebiasis is mainly due to extra-intestinal pathology, of which amoebic liver abscess (ALA) is the most common. If left untreated, ALA can rupture into neighboring tissue and spread to the brain and other organs via hematological route producing serious morbidity and mortality. It is difficult to differentiate clinically the ALA from pyogenic liver abscess (PLA) as well as from other space occupying lesions of liver such as hydatid cyst and liver hepatoma [2, 3].
Imaging techniques like ultrasound, computed tomography, and magnetic resonance although are highly sensitive to detect abscesses in the liver of varied aetiology, however fail to distinguish specifically ALA from that of PLA. Less than one third of patients with ALA have active diarrhea [3]. Hence, stool microscopy and stool antigen detection is not very helpful for diagnosis of ALA. In fact less than only 10% of ALA patients have identifiable E. histolytica in stool specimens [4].
Laboratory diagnosis of ALA is usually established by conventional antibody-based serological tests. Nevertheless, the main disadvantage with antibody detection is that serum antibody levels in individuals living in endemic areas, continues to remain positive even for years after infection with E. histolytica [5–7]. The demonstration of amoebic antibodies in the serum, therefore, fails to denote the amoebic infection whether it is recent or old. Furthermore, serum amoebic antibodies are not demonstrated in up to 10% of the patients with acute ALA [3].
The demonstration of E. histolytica trophozoite in liver abscess pus aspirates by microscopy confirms the diagnosis of ALA, but in best of the laboratories, the amoebic trophozoites can be demonstrated in only 15% of the liver pus [8]. Since the trophozoites of E. histolytica are found mainly in the periphery of the abscess diagnosis of ALA by culture of liver pus for E. histolytica is also unsatisfactory [9].
Demonstration of amoebic antigen in the liver pus is a recent approach for specific diagnosis of the ALA. A monoclonal antibody-based second generation TechLab enzyme-linked immunosorbent assay (ELISA) kit (Blacksburg, Va.) has been reported to be 78% sensitive for the detection of E. histolytica lectin antigen in the liver pus for the diagnosis of ALA in Dhaka, Bangladesh [10]. Studies conducted in various laboratories worldwide including ours have shown that polymerase chain reaction (PCR) is a sensitive and specific method for detecting Entamoeba DNA in stool samples and for differentiating the morphologically similar E. histolytica from Entamoeba dispar and Entamoeba moshkovskii [11–19]. However, only few studies using the PCR have been reported for the detection of Entamoeba DNA in liver abscess pus for the diagnosis of the ALA [20, 21, 9, 10].
Collection of blood or liver abscess pus is an invasive procedure, and the procedures require technical expertise and disposable syringes [22]. The method if not carried out under stringent conditions is associated with the risk of acquiring needle-borne infections such as hepatitis B virus and human immunodeficiency virus (HIV). Therefore, of late much interest has been shown towards the use of urine as a specimen alternate to the blood for the diagnosis of some parasitic infections including malaria, schistisomiasis, kala-azar, cystic echinococcosis and neurocysticercosis [22]. Urinary antigen for cystic echinococcosis (CE) and neurocysticercosis has been reported for the first time from our laboratory at Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India [23, 24]. Our laboratory has developed for the first time a counter-current immunoelectrophoresis (CIEP) and co-agglutination (Co-A) to detect the hydatid antigen excreted in the urine for the diagnosis of CE [23, 25], and Co-A to detect cysticercus antigen in the urine for the diagnosis of neurocysticercosis [24].
Detection of DNA in urine by PCR has been employed for the diagnosis of Toxoplasma gondii, Neisseria gonorrhoeae, Borrelia burgdorferi, Mycobacterium tuberculosis, Mycobacterium leprae and Chlamydia trachomatis infections [26–30]. Some studies have also shown that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies [31, 32]. To the best of our knowledge till now there is no report available on detection of Entamoeba DNA in the urine for the diagnosis of ALA. In the present study, therefore for the first time, we have evaluated a nested multiplex PCR for detection of Entamoeba DNA excreted in the urine for the diagnsosis of ALA.
The quantification of DNA in the liver abscess pus and urine specimen by Spectrophotometric analysis showed the DNA yield to be approximately 85 and 3 μg/ml respectively. The purity of DNA extract from liver abscess pus and urine specimens was found to be satisfactory as the value of ratio of readings at 260 nm and 280 nm (OD260/OD280) was approximately 1.8–1.85.
The sequencing result of PCR product of E. histolytica from liver abscess pus and urine specimen showed 99% identities to the sequence deposited in GenBank, [accession number: X56991]. The result of assessment of competition of other non target DNA present in liver abscess pus (PLA pus negative for E. histolytica) and urine (negative control group) specimen with target DNA, showed expected amplification and no nonspecific amplification in nested multiplex PCR.
Estimation of minimum number of Entamoeba cells detectable by nested multiplex PCR showed that the detection limit of PCR was found to be approximately 15 E. histolytica cells as even 1.5 μl of template DNA from 1000 E. histolytica cells/100 μl of Tris- ethylenediamine tetraacetic acid (EDTA) (TE) buffer produced a positive signal.
The IHA test was positive for serum antibodies in the serum of 86 (61.9%) of 139 patients provisionally diagnosed as ALA. The test was positive in a higher number of serum (71 of 102 [69.6%]) samples of patients who had received prior treatment with metronidazole than those who had not received any prior treatment with metronidazole (15 of 37 [40.5%]) and this difference was statistically significant (χ2 = 8.53, P = 0.003). Metronidazole treatment was initiated from a few days to several weeks before collection of the blood samples in these patients. Two (4.6%) out of 43 sera from control cases were positive for antiamoebic antibody by IHA.
The TechLab E. histolytica II test was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 56 (40.3%) of 139 provisionally diagnosed ALA patients. The TechLab E. histolytica II test detected lectin antigen in 30 (81%) of 37 liver abscess pus of patients which were collected prior to treatment with metronidazole. In contrast, the TechLab E. histolytica test detected the lectin antigen in only 26 (25.5%) of 102 liver pus (χ2 = 32.61, P < 0.001), collected after initiation of therapy with metronidazole. The probability of E. histolytica antigen detection in liver abscess pus by ELISA was found to be 12 times more in patients who had not received prior treatment with metronidazole (Odds Ratio (OR) = 12.53, 95% Confidence Interval (CI) = 4.55 to 35.86) than in the patients who received prior metronidazole therapy. The OR was statistically significant as the 95% CI of OR was more than 1.
Microscopy of the liver pus demonstrated E. histolytica trophozoites in 10 of 139 (7.2 %) liver abscess specimens, but only 2 (1.4%) pus specimens were positive by culture for E. histolytica. All 10 patients whose liver pus were positive for E. histolytica by microscopy and/or culture were also positive for E. histolytica Gal/GalNAc lectin antigen in the liver pus by the TechLab E. histolytica II test and serum amoebic antibodies by the IHA test.
A total of 102 out of 139 (73.4%) liver abscess pus were negative for aerobic bacteria by Gram's staining and bacterial culture. Twenty seven liver abscess pus specimens were positive for aerobic bacteria by Gram's staining and bacterial culture. These included Klebsiella pneumoniae (n = 9), Proteus species (n = 5), Enterobacter (n = 5), Escherichia coli (n = 3) and Pseudomonas (n = 5). Ten liver abscess pus specimens showed secondary infection of ALA with aerobic bacteria by Gram's staining and bacterial culture. These included K. pneumoniae (n = 3), Enterobacter (n = 2), E. coli (n = 1), group B Salmonella species (n = 1), Enterococcus (n = 1) and Coagulase negative Staphylococci (n = 2). Such secondary infection of ALA with bacteria has been reported previously in the literature [33, 34].
In the present study, a total of 112 out of 139 (80.6%) provisionally diagnosed ALA patients were diagnosed as ALA and remaining 27 patients were diagnosed as PLA, by following the criteria mentioned in this study elsewhere.
Comparison of result of PCR and antigen detection in liver abscess pus specimen of ALA patients.
PCR results
Antigen detection result (no. of specimens positive)
E. histolytica
Antigen negative
Total no. of specimens
E. histolytica
55 a
The nested multiplex PCR did not detect E. histolytica DNA in a total of 49 liver abscess pus specimens, which included 27 PLA and 22 ALA pus specimens. The probability of negative nested multiplex PCR results, in these 49 liver abscess pus specimens due to PCR inhibitors was ruled out by the inclusion of an internal amplification control (IAC) in the nested PCR reaction.
The comparison of results of nested multiplex PCR and TechLab E. histolytica II ELISA test on liver abscess pus from ALA patients is summarised in the table 1.
Detection of serum antiamoebic antibody, liver pus E. histolytica antigen, liver pus Entamoeba DNA and urine Entamoeba DNA in ALA patients.
No. (%) of positive results by:
IHA for :
ELISA for:
PCR for:
No of patients
Antiamoebic antibody in sera
Amoebic lectin antigen in liver pus
Entamoeba DNA in liver pus
Entamoeba DNA in urine
E. histolytica
E. dispar
E. moshkovskii
E. histolytica
E. dispar
E. moshkovskii
(439 bp)
(174 bp)
(553 bp)
(439 bp)
(174 bp)
(553 bp)
38 (71.7)
29 (54.7)
51 (96.2)
The nested multiplex PCR test detected E. histolytica DNA in the urine specimens of 4 (17.4%) of 23 ALA patients who were tested prior to treatment with metronidazole and in 17 (56.7%) of 30 ALA patients who were tested after treatment with metronidazole (χ2 = 6.83, P = 0.009). All of the 4 ALA patients, who did not receive prior treatment with metronidazole and whose urine specimens were positive for E. histolytica DNA, were available for follow-up study. Urine specimens were collected from these patients every week for 4 weeks after starting of the therapy with metronidazole; and were tested for E. histolytica DNA by PCR. It was observed that 2 weeks after treatment with metronidazole, 3 (75%) out of 4 urine specimens became negative for E. histolytica DNA. One urine specimen became negative for E. histolytica DNA after 4 weeks of treatment with metronidazole.
In this study, we have made an attempt to detect excretory E. histolytica DNA in urine by applying nested multiplex PCR and to assess the diagnostic potential of the test for detection of E. histolytica DNA in urine for the diagnosis of ALA. Also we have studied the kinetics of the excretion of E. histolytica DNA in urine during the course of therapy with metronidazole.
In our study, 76.8% (86 of 112) of ALA patients were positive for antiamoebic antibody in serum by the IHA. This result was similar to that reported from Bangladesh where serum antiamoebic antibodies were found in 78% of ALA patients [10], but differed from that of the study reported from South Africa, where serum antiamoebic antibodies were found in a higher 99% of ALA patients [35].
Only two out of 10 ALA pus samples which were positive for E. histolytica trophozoite by microscopy were positive for the amoebae by culture and the rest were negative, this may be due to the inhibition of growth by culture itself. In majority of patients, K. pneumoniae was the major bacterial pathogen responsible for PLA and as secondary bacterial infection of ALA. One of 10 ALA pus specimens was positive for Group B Salmonella species, this patient had liver abscess with perihepatic collection, with severe sepsis and disseminated intravascular coagulation, finally the patient died. In this study, the anaerobic culture of liver abscess pus aspirate was not done. Therefore, the possible etiology of liver abscess due to anaerobic organisms such as Bacteroides remained undetermined.
In the present study, 50% (56 of 112) of liver abscess pus were positive for E. histolytica lectin antigen. The sensitivity of the test in our study was observed to be slightly higher than that of the study using the same TechLab E. histolytica II kit (40.7 % sensitiivty) on liver pus reported from Bangladesh [10]. However, results of other studies using polyclonal antibody based ELISA showed a very high sensitivity for the detection of amoebic antigen in the liver pus. Amoebic antigen was detected in liver abscess pus in 97.6% (41/42) of ALA cases by ELISA as reported from China [21] and in 92% and 96% of liver pus by using immunoelectrophoresis and ELISA respectively, from India [36].
In developing countries like India where amoebiasis is endemic, antiamoebic drugs and antibiotics are used indiscriminately, making it difficult to obtain an accurate treatment history. Most of the patients in the present study had already been treated with metronidazole at the time of collection of clinical specimens. The serum amoebic antibodies were detected in higher percentage (94.7%) of ALA patients treated earlier with metronidazole, but were detected in only 40.5% of patients who did not receive any prior treatment with metronidazole. This might be due to the late antibody response during the course of the disease.
Unlike serum amoebic antibody detection, E. histolytica lectin antigen was detected in liver pus by TechLab ELISA in a higher percentage (81%) of ALA patients, who were tested prior to treatment with metronidazole, but was detected in only 34.6% of ALA patients, who were tested after the initiation of therapy with metronidazole. This might be due to the rapid clearing of amoebic antigen from the liver pus due to killing of E. histolytica trophozoites on treatment with metronidazole.
The PCR for the detection of E. histolytica DNA in liver abscess pus had a much higher sensitivity (100%) when tested prior to treatment with metronidazole, but had a lower sensitivity (70.6%) when tested after the initiation of treatment with metronidazole. This might be attributed to the clearing of E. histolytica DNA from the liver abscess due to the death and lysis of E. histolytica trophozoites on treatment with metronidazole. The percentage of agreement between E. histolytica DNA detection and ELISA for E. histolytica antigen detection in liver abscess pus was found to be 67.9% in the present study (Table 1). The Kappa statistic was found to be 0.36 which indicates a fair agreement between the two tests.
PCR for detection of E. histolytica DNA and ELISA for detection of E. histolytica lectin antigen in liver abscess pus were evaluated for the diagnosis of ALA (McNemar's χ2 = 30.25, p < 0.0001). The sensitivity of PCR was 80.4%. This was found to be significantly higher than that of ELISA (50 %) using McNemar's χ2 test (p < 0.0001). All 27 liver abscess pus aspirates diagnosed as PLA were negative for E. histolytica DNA by PCR and for E. histolytica lectin antigen by TechLab ELISA, which represents a specificity of 100%. ELISA for detection of liver abscess pus E. histolytica lectin antigen demonstrated a 100% positive predictive value and a 32.5% negative predictive value. PCR for the detection of liver abscess pus E. histolytica DNA demonstrated a 100% positive predictive value and a 55.1% negative predictive value.
In the present study E. histolytica DNA was detected in the urine specimen of 4 (17.4%) of 23 ALA patients, who were tested prior to treatment with metronidazole and in 17 (56.7%) of 30 ALA patients, who were tested after treatment with metronidazole by PCR. The probability of E. histolytica DNA detection in urine by PCR was 6 times more in ALA patients who had received prior metronidazole therapy (OR = 6.2, 95% CI = 1.47 to 28.37) than in the ALA patients who did not receive prior metronidazole therapy. The OR was statistically significant as the 95% CI of OR was greater than 1. This might be due to release of increased E. histolytica DNA from the dying E. histolytica trophozoites when metronidazole therapy was initiated, leading to excretion of E. histolytica DNA in the urine. One study has demonstrated that the DNA from dying cells can cross the kidney barrier in rodents and humans and can get excreted with urine, which can be used for genetic analysis [31].
PCR for detection of E. histolytica DNA in liver abscess pus and urine specimen were evaluated for the diagnosis of ALA (McNemar's χ2 = 26.28, p < 0.0001). The sensitivity of PCR for urine was 39.6%. This was found to be significantly lower than that of PCR for liver abscess pus (80.4%) using McNemar's χ2 test (p < 0.0001). All urine specimens from 15 PLA patients and 43 control group individuals were negative for E. histolytica DNA by PCR. This represents a specificity of 100% (Table 2). PCR for the detection of urinary E. histolytica DNA demonstrated a 100% positive predictive value and a 31.9% negative predictive value.
As per the PCR kinetics the likelihood of amplifying smaller PCR product is more than amplifying larger PCR product. We feel that if the PCR product smaller than 400 bp would have been amplified, the PCR might show higher sensitivity.
E. histolytica DNA in urine did not persist longer in ALA patients after treatment with metronidazole as observed in the present study. Three of 4 (75%) urine specimens positive for E. histolytica DNA became negative for E. histolytica DNA within 2 weeks of treatment with metronidazole. This might be attributed to the reduced excretion of E. histolytica DNA in the urine as a result of reduction of E. histolytica infection load following treatment with metronidazole. The effect of metronidazole in killing of E. histolytica and clearing of antigenemia in hamsters suffering from hepatic amoebiasis has been well documented in a study reported by Thammapalerd et al. [37] Results of the present study therefore indicate that the PCR can be used to monitor excretion of E. histolytica DNA in urine as a prognostic marker during therapy of ALA with specific antiamoebic drugs.
In the present study, none of the liver abscess pus and urine PCR results were positive for either E. dispar or E. moshkovskii specific PCR products, which confirm the non invasive nature of these species (Table 2).
The detection of E. histolytica DNA and E. histolytica specific lectin antigen in the serum specimen of ALA patients was not carried out in this study. A controlled prospective study to evaluate the detection of E. histolytica DNA and E. histolytica specific lectin antigen in the serum specimen of ALA patients has been intended to be carried out in future in our laboratory.
The present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica DNA in urine of patients with ALA can also be used as a prognostic marker to assess the course of the diseases following therapy by metronidazole. The detection of E. histolytica DNA in urine specimen of ALA patients provides a new approach for the diagnosis of ALA.
Sample details
The subjects in the present study included 139 patients provisionally diagnosed as ALA, who were admitted to JIPMER hospital, Puducherry, as well as 43 controls during a period from September 2004 to March 2006. The provisional diagnosis of ALA was made by the physicians on the basis of patient's history and clinical features, unfortunately these features are often nonspecific to confirm the diagnosis of ALA. Of the 139 provisionally diagnosed ALA patients, 102 had received prior treatment and 37 did not receive prior treatment with metronidazole. The patients and controls were residing in neighboring area of Puducherry. Informed consent was obtained from the patients. This study has been performed with the approval of Institute Research Council of JIPMER, Puducherry.
The control group included 43 individuals who had no history of recent dysentery or diarrhea and whose stool samples were negative for E. histolytica infection by microscopy and culture. Thirty five of the controls were healthy asymptomatic volunteers, and the other 8 patients included, hydatid cyst in liver (n = 2), liver hepatoma (n = 1), liver cirrhosis (n = 3), and viral hepatitis (n = 2).
The diagnosis of ALA was established on the basis of radiological, symptomatological and laboratory criteria as follows [3, 10]: (i) a space-occupying lesion in the liver diagnosed by ultra sonography and suggestive of abscess, (ii) clinical symptoms (fever, pain in the right hypochondrium (often referred to the epigastrium), lower chest, back, or tip of the right shoulder), (iii) enlarged and/or tender liver, usually without jaundice, (iv) raised right dome of the diaphragm on chest radiograph, (v) improvement after treatment with antiamoebic drugs (e.g., metronidazole) (vi) positive IHA of serum antibody showing a titer (≥ 1:128) against E. histolytica, (vii) liver aspirate appeared like anchovy sauce but was bacteriologically sterile.
Sample collection
Liver abscess pus: The aspiration of liver abscess pus was indicated only under the following conditions [3]. (i) to rule out a pyogenic abscess; (ii) the failure to respond clinically in 3 to 5 days; (iii) the threat of imminent rupture; and (iv) the prevention of rupture of left-lobe abscess into the pericardium. The liver abscess pus aspirates were performed, only for clinical purposes as judged by the clinicians for the patient care and not for the purpose of this study. Liver abscess pus was obtained under ultrasound guidance from all 139 provisionally diagnosed ALA patients and was stored at -20°C in a sterile container until used.
Urine: Urine specimen could be collected from 68 out of 139 provisionally diagnosed ALA patients and all 43 control group individuals included in the study. 10 ml of urine specimen was collected in a sterile container using aseptic techniques; urine sample was stored at -20°C until use.
Blood: Blood specimen was collected from all 139 provisionally diagnosed ALA patients and 43 control group individuals included in the study. Venous blood (5 ml) was collected in a sterile container; sera were separated and stored at -20°C until used.
Microscopy for Entamoeba
Liver abscess pus: The specimens were examined immediately after the aspiration of abscess. The liver abscess pus was first centrifuged at 2,500 g for 5 mins and a loopful (usually inoculating needle loop) of sediment was mixed with a drop of warm saline on a microscope slide. Microscopic examination of an amoebic abscess aspirate from liver may reveal haematophagous trophozoites.
Culture for Entamoeba
Liver abscess pus: Liver abscess pus specimens were cultured for Entamoeba species in Locke-egg (LE) medium (NIH modification of Boeck and Drbohlav's medium) as previously described [38]. The liver abscess pus was first centrifuged at 2,500 g for 5 mins and a loopful of sediment was inoculated into the LE medium. It is to be noted that in case of culturing Entamoeba from liver abscess aspirates, since the abscess is sterile, bacterial flora (ATCC E. coli) was added before inoculation of amoebae into xenic culture.
Gram stain and bacterial culture for liver abscess pus aspirates
Direct smear Gram staining and bacterial culture was done for all liver abscess pus aspirates. The liver abscess pus specimens were inoculated on to sheep blood agar, MacConkey agar and chocolate agar plates. The MacConkey agar plates were incubated at 37°C for 18–24 hours whereas the blood agar and chocolate agar plates were incubated in a candle jar at 37°C for 48 hours. Based on the colony morphology and result of culture smears, necessary biochemical tests were done to identify bacteria to the species level.
TechLab E. histolytica II ELISA test
The TechLab E. histolytica II test was performed on liver abscess pus specimens to detect E. histolytica as per the method described earlier [10].
Antiamoebic antibody detection by rapid-IHA test
The Rapid-IHA was performed on serum specimen as per the method described earlier [39]. Briefly, double aldehyde stabilized chick red blood cell (RBC) sensitized with the amoebic antigen was used in the test. The haemagglutination test was performed on test serum samples including known positive and negative control sera in each batch. The chick RBC settled quickly and their haemagglutination pattern could be determined within 30 to 45 min of incubation at room temperature with test sera [39]. A titer of ≥ 1:128 was considered positive for ALA [40].
Extraction of Entamoeba genomic DNA
Liver abscess pus: Briefly, 1 ml of liver abscess pus was taken in 1.5 ml centrifuge tube and centrifuged at 12, 000 g for 8 minutes. The supernatant was discarded and 50 μl of pellet was dispersed in 250 μl of lysis buffer (0.25% sodium dodecyl sulfate (SDS) in 0.1 M EDTA, pH 8.0), followed by addition of 100 μg/ml of proteinase K. The lysate was incubated at 55 °C for 2 hours. Then 75 μl of 3.5 M sodium chloride (NaCl) followed by 42 μl of 10% cetyltrimethylammonium bromide (CTAB)/0.7 M NaCl (heated to 55 °C) was added. After the components were mixed, the sample was incubated at 65°C for 30 min. This was followed by extractions with equal volumes of chloroform and then phenol-chloroform-isoamyl alcohol, and the DNA was precipitated with ice cold ethanol. The dried DNA pellet was dissolved in 50 μl of sterile distilled water.
Urine: Briefly, 10 ml of the urine sample was centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was discarded and the pellet was suspended in 500 μl of sterile distilled water. The suspension was boiled for 10 minutes followed by sudden cooling. Next, 5 μl of proteinase-K (10 mg/ml) and 60 μl of 10% SDS were added to the suspension and incubated at 65 °C for three hours. Then 80 μl of 5 M NaCl and 20 μl of 10% CTAB were added to the mixture and incubated at 65 °C for 45 min. This was followed by extractions with equal volumes of chloroform and then phenol-chloroform-isoamyl alcohol. The DNA was precipitated with ice cold ethanol. The dried DNA pellet was dissolved in 50 μl of sterile distilled water.
The protocol for extraction of DNA from liver abscess pus and urine specimen has been modified in our laboratory from CTAB DNA extraction protocol originally described for DNA extraction from amoebic culture [41]. The extracted DNA from liver abscess pus and urine sample was passed through DNA clean-up spin columns (Bangalore Genei KT-62, Bangalore). The DNA was stored at -20 °C until used.
Quantification of DNA in liver abscess pus and urine specimen
DNA quantification in spin column purified DNA extract from liver abscess pus and urine specimen was determined by UV absorbance using a Cintra 5 double beam Spectrophotometer. DNA yields were calculated on the basis of UV absorbance × dilution. The purity of the nucleic acid in the samples was estimated by the ratio of readings at 260 nm and 280 nm (OD260/OD280).
Primers design
The genus specific primers were designed using nucleotide sequences of 16S-like rRNA gene of E. dispar, E. histolytica and E. moshkovskii Laredo deposited in GenBank [accession number : Z49256], [accession number : X56991] and [accession number : AF149906] respectively.
Primer sequence used in PCR.
Genus specific primers (First PCR)
Entamoeba genus
E-1 5' TAAGATGCACGAGAGCGAAA 3' (forward primer)
E-2 5' GTACAAAGGGCAGGGACGTA 3' (reverse primer)
Species specific primers (Second nested multiplex PCR)
E. histolytica species
EH-1 5' AAGCATTGTTTCTAGATCTGAG 3' (forward primer)
EH-2 5' AAGAGGTCTAACCGAAATTAG 3' (reverse primer)
E. moshkovskii species
Mos-1 5' GAAACCAAGAGTTTCACAAC 3' (forward primer)
Mos-2 5' CAATATAAGGCTTGGATGAT 3' (reverse primer)
E. dispar species
ED-1 5' TCTAATTTCGATTAGAACTCT 3' (forward primer)
ED-2 5' TCCCTACCTATTAGACATAGC 3' (reverse primer)
Internal amplification control (IAC) primer for PCR
Human 18S ribosomal RNA gene
IAC-1 5' GGCTTTGGTGACTCTAGATA 3' (forward primer)
IAC-2 5' CGTTAAAGGATTTAAAGTGG 3' (reverse primer)
Standard strains
Three standard strains used in this study were E. histolytica HM-1: IMSS, E. dispar SAW760, and E. moshkovskii Laredo these were used as positive control in the present study. The lyophilized DNA of these strains was generously gifted by Dr. C. Graham Clark from London School of Hygiene & Tropical Medicine, London, UK.
Nested multiplex PCR protocol
Liver abscess pus PCR: For a reaction volume of 25 μl, comprising 2.5 μl of 10X PCR buffer (Biogene), 2.0 μl of 25 mM MgCl2 (Bangalore genei), 0.75 μl of deoxynucleoside triphosphate mix (10 mM each dNTP, Biogene), 0.3 μl (5 IU/μl) of Taq polymerase (Biogene), 10 picomoles of target DNA primers (IDT) and 5 picomoles of IAC primers (IDT) were added in genus and species specific PCR. The template DNA volume was 2 μl for both genus and species specific PCR. The PCR tubes were finally placed in an Eppendorf Thermal cycler [Master cycler gradient].
Urine PCR: The PCR mix composition was the same as described earlier for liver abscess pus PCR, except that 1.0 μl of 25 mM MgCl2 and 2.5 μl of template DNA was added.
The conditions for genus specific PCR were as follows; the PCR mix was subjected to an initial denaturation at 96 °C for 2 minutes, followed by 30 cycles – each consisting of 92 °C for 60 seconds (Denaturation), 56 °C for 60 seconds (Annealing), and 72 °C for 90 seconds (Extension). Finally one cycle of extension at 72 °C for seven minutes was performed. In the species specific nested multiplex PCR (which had multiple primer sets in the same tube), only the annealing temperature was changed to 48 °C, leaving the other parameters of the amplification cycles unchanged.
3.5 μl of the amplification product was separated by electrophoresis through 1.8% agarose gel (Agarose Low EEO, Bangalore genie products, Bangalore, India) containing ethidium bromide in 0.5 × Tris-borate-EDTA at 120 V for 45 min and was visualized under UV light for bands of DNA of appropriate sizes (Figure 1). A negative control reaction was included with each batch of samples analyzed by PCR.
Primer validation
The primer sequences designed for E. moshkovskii, E. histolytica, E. dispar and Human IAC were subjected to Basic Local Alignment Search Tool (BLAST) in the genome database of all organisms [43] and were found to be specific for the study. The amplified PCR products of E. histolytica species in liver abscess pus and urine samples was confirmed by getting both the strands of DNA sequenced on ABI3730XL sequencer (Macrogen, Seoul, South Korea). The sequencing was done using species specific primers i.e. EH-1/EH-2 for E. histolytica. All sequences were analyzed for homology by using the nucleotide-nucleotide BLAST search feature [43].
The identity between the sequencing results of PCR product of E. histolytica from liver abscess pus and urine with the sequence deposited in GenBank [accession number: X56991] were analyzed by using the "Align two sequences (bl2seq)" feature [43].
Assessment of competition of non target DNA
During the standardization to assess the competition of other non target DNA present in liver abscess pus and urine specimen with target DNA, the nested multiplex PCR was checked with reference DNA (DNA from standard culture of E. histolytica, E. dispar &E. moshkovskii) spiked with DNA from liver abscess pus (PLA pus negative for E. histolytica) and urine (negative control group) followed by nested multiplex PCR amplification.
Estimation of minimum number of Entamoeba cells detectable by nested multiplex PCR
This was performed for E. histolytica with Locke-egg (LE) medium (NIH modification of Boeck and Drbohlav's medium) liver abscess pus cultures; the amoebae were counted using a standard haemocytometer. A cell pellet containing 106 cells was preferred for determining the detection limit of nested multiplex PCR for E. histolytica. The cell pellet containing 106 cells of E. histolytica was diluted 10 folds in phosphate buffer saline (PBS) to obtain different concentration of cells, such as 105, 104, 103, 102 and 10 cells/ml. The different dilutions of cells ranging from 106 to 10 cells/ml were centrifuged and the remaining pellet of each dilution was added to 0.05 μl of liver abscess pus (PLA pus negative for E. histolytica) followed by DNA extraction and PCR as per the aforementioned protocol.
Statistical data analysis
Sensitivity was calculated as follows: number of patients with positive test results/total number of patients × 100. Specificity was calculated as follows: number of controls with negative test results/total number of controls × 100. The positive predictive value was calculated as follows: number of true positives/(number of true positives + number of false positives) × 100. The negative predictive value was calculated as follows: number of true negatives/(number of true negatives + number of false negatives) × 100. The agreement between the tests was calculated using the Kappa statistics. To determine the statistical significance of differences between the proportions, Chi-square (χ2) test and Fisher's exact test were used. The χ2 test and odds ratio were found using "Epi Info Version 6". To calculate the significance of the difference in sensitivities, McNemar's Chi-square test was applied. The McNemar's test was performed using "Graph Pad Software".
We sincerely thank Dr. K.V. Kirubasankar and Dr. Gowrisankar from Department of Preventive and Social Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry for doing the statistical data analysis for this work.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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After a sell out gig last year, China Crisis are returning to Thornton Little Theatre for one night only.
And tickets have already gone on sale for the October 1 gig, with demand expected to be high.
It was a great crowd at our gig last year and we are very much looking forward to returning to this wonderful little venue in Thornton
Formed in Liverpool in 1979 by Gary Daly and Eddie Lundon, the two started working together after leaving school.
Now, with a string of top 40 singles and albums to call upon as well as new music, this will be a night that will be loved by fans new and old.
Original members, Eddie and Gary will be playing all the familiar songs including Black Man Ray and Wishful Thinking.
Gary said: "It was a great crowd at our gig last year and we are very much looking forward to returning to this wonderful little venue in Thornton.
"We love being on tour and playing at intimate venues, it's always good to see some familiar faces in the crowd and to play to new fans."
Tickets are £16 and are selling fast for the intimate gig, with fans urged to buy now to avoid disappointment.
To book tickets, call the Wyre Theatres box office on (01253) 887693 or visit www.marinehall.co.uk for a link.


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12 October 2009
Australia's Alice Springs named as haven for lesbians
Anyone looking for a friendly lesbian town - male or female - has been encouraged to visit Alice Springs in the Northern Territory: the lesbian capital of Australia. Lesbians are also encouraged to emigrate to Australia, with Australian immigration law recognising same-sex couples.
Tourism Australia Northern Territory regional director Phil Walcott told the NT News that Alice Springs is the undiscovered lesbian capital of Australia.
Mr Walcott said Alice Springs had a high per-capita residency of lesbians, and a recent documentary shown on Australian television, Destiny in Alice, made a light-hearted exploration of the small town's lesbian lifestyle.
Alice Springs, in the Northern Territory, became somewhat of a haven for lesbians after several hundred women took part in a march protesting against the Pine Gap intelligence base in 1983.
The documentary explained that many participants in the protests were lesbians who came, saw and decided to stay.
An Australian spouse and partner visa can be applied for by the same sex partner of an Australian citizen or Australian permanent resident who wishes to emigrate to Australia to be with their partner.
To visit Alice Springs or anywhere else in Australia all visitors, unless they have an Australian or New Zealand passport, must have an Australian tourist visa. The most popular Australian tourist visa is the ETA visa.
Click here to Apply Online for your ETA Tourist Visa
The Australian Visa Bureau is an independent consulting company specialising in helping people emigrating to Australia and their Australia visa applications.


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The first one I did was Crockpot Lemon Chicken; I found the receipe through Cheap Meals for your Family, this is a GREAT site for recipes. The recipe did have a link to Annie's Recipes, I presume this is the orignator of the recipe.
Prep Time: 10 minutes (this is for seasoned cooks, it took me a bit longer)
Cook Time: 7-9 hours low or 4-5 hours high (I used high setting 6 hours on my crockpot)
Meal Preparation Ingredients:
1 pkg whole chicken cut up (buying your chicken like this is very cheap)
1/4 cup of flour
1 1/2 tsp. salt
2 Tbsp. oil
1 (6oz.) can lemonade, thawed and undiluted
3 tbsp. brown sugar
1 tbsp vinegar
1/4 c catsup
Meal Preparation Directions:
Combine flour and salt and coat chicken. Brown on all sides in hot oil. Place browned chicken in crockpot. Combine lemonade, vinegar, catsup and brown sugar, mix well and pour over chicken. Cook on low 6-8 hours or high 3-4 hours.
My notes: I am not as much of a seasoned cook as I would like to be, so it did take me more than 10 minutes to prepare. I used a whole cut chicken, plus two additional chicken breast (rib meat and bone in). I seasoned the meat and the flour with complete season salt rather than just salt (I'm Puerto Rican, I need flavor). I made rice to serve on the side with green beans. Overall VERY easy, and delicious!The second recipe was also found at Cheap Meals for your Family, it was Chicken and Rice Bake. This was especially great (and easy) because it was a one dish meal. I prepared and baked in the same dish.
Chicken and Rice bake:
1 lb of chicken (I used boneless/skinless breast for this recipe)
3/4 cup of uncooked white rice, not instant rice
1 can cream of mushroom soup
1/4 tsp paprika
1/4 tsp black pepper
1 1/3 cup of water
1 can green beans
Stir water, rice, soup and seasoning in shallow baking dish then add chicken. Sprinkle chicken with additional seasoning. Bake at 375 for 45 min to 1 hour or until rice and chicken are done. As the picture shows you can top the chicken with cheese and toss back in the oven for about 5 minutes to melt. Serve with green beans. Feeds 4-5 people.
My notes: I did not deviate from this recipe much, except of course I used more seasonings on the chicken. Additionally, although there was rice in the baked dish, I did make more on the side.I thank those who had already shared these recipes so that I could find them to use! I hope this info is beneficial to some of you as well.


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I mentioned, on sister site Croatia Online (COL – Crikvenica), that part of Crikvenica looked as if it might be in need of a little TLC and it seems, on further research, that investment might be just around the corner. It's already got plenty to offer, including sandy beaches and all the essential amenities and facilities, but is a little rooted in an almost bygone age of tourism.
For nautical visitors to Croatia it also already offers quite a lot including a fuel berth, pictured, and several smallish harbours, mostly for local and tripper boats. Soon, however, it may get its own 200 berth marina and we'll be telling you more about that in due course. In the meantime they are a few options for visiting yachts:
a) in the main harbour, just west of the river mouth, berth on lazy lines on the inner side of the main breakwater and east harbour wall in depths of around 3 metres but note that the outer end of the jetty is mainly for tripper boats and the inner harbour for local boats
b) there are rings and bollards for lines along the quay by the river mouth. Depths vary between 2 and 4 metres and there's a low road bridge just a little way down river
c) a little further east there's Luke Podvorska, the newish harbour for local boats but that's usually full
The main harbour has some smart new electricity and water pedestals and there's a green light at the end of the long breakwater with another green light further east on the west side of the river mouth. The fuel station is immediately west of this second green light and has depths of approximately 3 metres.


text: Irish writer/director Johnny O'Reilly, who lived in Moscow for twelve years, has made a compelling Russian drama telling the story of five interlocking characters on a single day, the Moscow City Day holiday, celebrated with parades and fireworks. Like a Russian Crash, the film dives headlong into the volatile intersections and intimate lives of five people. A high-powered entrepreneur is threatened with losing everything in a fight with corrupt government officials. His beautiful, mercenary singer girlfriend is set to perform in a large outdoor concert that night, but is secretly being wooed by her lovelorn ex-boyfriend. A famous former film star, aging and critically ill, just wants to find a drink, but instead gets embroiled in a bizarre kidnapping. Featuring a strong cast and beautiful vistas of the Moscow cityscape, Moscow Never Sleeps reveals the unrestrained energy of Europe's biggest city and the savagery and beauty of the Russian spirit. The film stars many of Russia's best-known actors, including Aleksey Serebryakov (Leviathan). (Fully subtitled)


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Scraping Through
Strapping on my air guitar and thrashing it out on stage to Powderfinger was the ultimate thrill. Not to mention presenting the weather for the six o'clock news in my rock chick getup.
I also had the pleasure of watching Adam perform in Albury last week. He is such a natural performer and it was great to see him on stage. When he invited me up to sing, the audience made me feel very welcome so I felt quite relaxed.
We sang one of my all time favourites and I felt spoilt having the opportunity to sing it on stage with Adam. Next week you'll see us perform it again for the country genre – I can't wait!
My nerves weren't as bad last week so hopefully that'll only improve. Although I have to admit the elimination process was very nerve-wracking, especially when we were the last three on stage. I thought it was all over.
Several thoughts flashed through my mind. Firstly, what a shame Adam and I wouldn't have the chance to perform country given it's his thing. Secondly, my Mum (who lives in Dubai) had just emailed to tell me she was flying over to watch me perform the following week. And thirdly, I was having too much fun to leave!
Thankfully we made it through and I think last week taught me a good lesson. Obviously song choice is really important and even though I love Powderfinger, the song didn't really give me the chance to show off much vocal range. All that thrashing on stage also made it quite hard to sing!
Next week's song is simple, but incredibly beautiful and emotional. Every time I hear it I feel her pain and that's what I hope to portray. This Tuesday it's all about the music.


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You are currently browsing the tag archive for the 'newborn procedures' tag.
Trying to decide about giving your newborn Vit K at birth? This is an interesting argument.
Vitamin K – An Alternative Perspective.
Newborns are routinely given erythromycin as a prophylaxis to prevent ophthalmia neonatorum, an eye infection usually caused by chlamydia and gonorrhea. Read about the risks and benefits of this routine procedure and make an educated decision on if it's something you would like to decline for your baby.
This is just such a beautiful video – such a respectful and gentle bath for this new soul. What if we were all treated this way at birth?
If you're choosing to have your baby in the hospital, it's important to understand what newborn procedures are done and why so you can make a fully informed choice as to whether you want to have them done or would like to decline them.


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2018 Induction Ceremony Ticket On Sale & Rock Week Details
There are plenty of once-in-a-lifetime experiences
happening right here in the birthplace of rock 'n' roll, Cleveland, April 7-14, including the opening of the all new Hall of Fame Inductee Experience, Induction Ceremony and official Simulcast.
Tickets for the Induction Ceremony are now sold out.
The Ceremony will take place on Saturday, April 14 at Public Auditorium in Cleveland. It will honor this year's inductees: Bon Jovi, The Cars, Dire Straits, The Moody Blues, Nina Simone, and Sister Rosetta Tharpe.
To celebrate the class of 2018, the City of Cleveland, Inductees, partners, fan clubs, and local organizations are helping the Rock Hall amplify its mission to engage, teach and inspire through the power of rock and roll.
Rock Week highlights include:
- New grand opening of an all-new Hall of Fame Experience (level 3)
- Dozens of local restaurants, music venues and shops hosting Induction-themed events and specials during the week.
- Rock 'N Blast fireworks show
- Official Simulcast at the Rock Hall
- Live music and performances at the Rock Hall and throughout the City.
- Added Stories of Rock, education programs, films and more to enhance your Museum visit.
- And, of course, the Induction Ceremony with a star-studded red carpet
Follow @rockhall or signup for the Rock Hall newsletter to find out about added events, special guest appearances, and more.
Tickets to the official Simulcast are now sold out. The Simulcast will be held at the Rock Hall on Saturday, April 14 with doors opening at 6:30 pm.
Make your travel reservations for Induction Week now. Purchase your Rock Hall admission tickets in advance online and save.
While Cleveland isn't a place for people who follow the herd, it is for those who know how to have a good time. It's what happens when you never take yourself too seriously.
Find out more things to do in Cleveland HERE.
The return of the ceremony to Cleveland would not be possible without generous support from sponsors and supporters, including PNC Bank, Destination Cleveland, City of Cleveland, the State of Ohio, J.P. Murphy Foundation, Medical Mutual of Ohio, Greater Cleveland Partnership, Downtown Cleveland Alliance, Forest City Charitable Enterprises, and many other generous supporters.


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Subscribe To Why Bill & Ted 3 Will Be Even Funnier, According To Alex Winter Updates
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Some actors have a fear of being typecast. Locked into a role they played that seems to have defined them forever. Luckily for Alex Winter, forever being Bill S. Preston ESQ. of the Bill & Ted movies isn't a problem for him. He's actually really looking forward to having a chance to play the role again. He says the fact that it's been more than 25 years since the first film is what's going to make the third film great.
Speaking with The Next Web, Winter says that although he is older, Bill is not, and that's the joke:
It's exciting to come back to the characters. The fun thing about them is that they're ageless. You play them in terms of who they are spiritually. Their mannerism and physicality are not tied to age. We played them like 9-year-old kids. They still have that quality. The comic potential of that becomes even better as it becomes more discordant. It will be funnier because they're older.
If it's funny to see high school kids act like children, seeing grown adults do it will be even funnier. Could work. While a third Bill and Ted movie could run the risk of being a little too silly, at the same time, what else would you expect to see? Still, it's been a long time. It would be one thing if there had been several Bill & Ted movies over the last quarter century to keep the characters fresh, but after 25 years (How old were you in 1989?) seeing the characters again may be a bit of a shock to the system. Are Bill & Ted ageless, or were they a part of their own time? If done right, it doesn't really matter. Discordant is a good word for it. When Keanu Reeves started taking more serious acting roles he got a lot of flack because fans only knew him as "Ted." Now imagine the guy who was in the Matrix trilogy and John Wick going back and playing Ted. As Ted himself would say, "Whoa."
Bill & Ted 3 is one of those many projects that has been dancing through development hell for years. It's on. No, it's off. Well maybe. Who knows. It's been a few years since we've heard anything concrete, at one point the film had a finished script and a director attached but that was a while back, so it's possible a lot has changed. However, it does look like the film is on a slow and steady pace to being a real thing. At least at the moment.
I'm really not sure if it will work, but if Bill & Ted 3 ever happens you can bet I'll be in the theater to find out.