Ten Years of PubMed Central: a Good Thing that’s Only Going to Get Better.

26 05 2010

PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH’s National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM) (see PMC overview).
PMC is a central repository for biomedical peer reviewed literature in the same way as NCBI’s GenBank is the public archive of DNA sequences. The idea behind it “that giving all users free access to the material in PubMed Central is the best way to ensure the durability and utility of the electronical archive as technology changes over time and to integrate the literature with other information resources at NLM”.
Many journals are already involved, although most of them adhere to restrictions (i.e. availability after 1 year). For list see http://www.ncbi.nlm.nih.gov/pmc/journals/

PMC, the brain child of Harold Varmus, once the Director of the National Institutes of Health, celebrated its 10 year anniversary earlier this year.

For this occasion Dr. Lipman, Director of the NCBI, gave an overview of past and future plans for the NIH’s archive of biomedical research articles. See videotape of the Columbia University Libraries below:

more about “Ten Years of PubMed Central | Scholar…“, posted with vodpod

The main points raised by David Lipman (appr. time given if you want to learn more about it; the text below is not a transcription, but a summary in my own words):

PAST/PRESENT

  • >7:00. BiomedCental (taken over by Spinger) and PLoS ONE show that Open Access can be a sustaining way in Publishing Science.
  • 13:23 Publisher keeps the copyright. He may stop depositing but the content already deposited remains in PMC.
  • 13:50 PMC is also an obligatory repository for author manuscripts under various funding agencies mandates, like the NIH and the UK welcome trust.
  • 14:31 One of the ideas from the beginning was to crosslink the literature with the underlying molecular and other databases. For instance NCBI is capable of mining out the information in the archived text and connecting it to the compound and the protein structure database.
  • 16:50 There is a back issue digitization for the journals that are participating, enabling to find research that you wouldn’t have easily found otherwise.
  • PMC has become international (not restricted to USA)
  • The PMC archive becomes more useful if it becomes more comprehensive
  • Before PMC you could do a Google Scholar search and find a paper in PubMed, that appeared funded by NIH, but then you had to pay $30 for it in order to get it. That’s hard to explain to the taxpayers (Lipman had a hard time explaining it to his dad who was looking for medical information online). This was the impetus for making the results of NIH-sponsored results freely available.

PRESENT/FUTURE

  • 23:00 Discovery initiative: is the use of tracking tools to find out which changes to the website work for users and which don’t. Thus modifications should lead to alterations in users behavior (statistics is easy with millions of users). Discovery initiative led to development and improvement of sensors, like sensors for disease names, drug names, genes and citations. What is being measured is if people click through (if it isn’t interesting, they usually don’t) and how quickly they find results. Motto: train the machine, not the users.
  • 30:37 We changed the looks of PMC. Planning to make a better presentation on the i-phone and on broad monitors.
  • 31:40. There are almost 2 million articles in PubMed Central, 585 journals fully participate in PMC
  • 32.30 It takes very long to publish a paper, even in Open Access papers. Therefore a lot of people are not publishing little discoveries, which are not important enough to put a lot of time in. Publishing should be almost as easy as writing a blog, but with peer review. This requires a new type of journal, with peer review, but with instant feedback from readers and reviewers and rapid response to comments. The Google Knol authoring system offers a fast and simple authoring system where authors (with a Google profile) can collaborate and compose the article on the server. Uploading of documents and figures is easy, the article updates are simple and fast, there is a simple workflow for moderators. After the paper is accepted you press a button, the paper is immediately available and the next day PMC automatically gets the XML content. There is also a simple Reference Manager included to paste citations.
  • Principle: How you can start a journal with this system (see Figure). Till now: 60 articles in PLOS Currents Influenza. There are also plans for other journals: the CDC is announcing a Systematic Reviews journal, for instance.

QUESTIONS (>39:30):

  • Process by which “KNOL-journal” is considered for inclusion in NLM?
    • Decide: is it in scope?, implicit policy (health peer review being done), who are the people involved, look at a dozen articles.
  • As the content in PMC increases, will it become possible to search in the full text, just like in Google Scholar?
    • Actually the full text is searchable in PMC as apposed to PubMed, but we are not that happy with the full text retrieval. Even with a really good approach, searching full text works just a little bit better than searching PubMed.
      We are incorporating more of the information of PMC into PubMed, and are working on a separate image database with all the figures from books and articles in PMC (with other search possibilities). Subsets of book(chapter)s (like practice guidelines) will get PubMed abstracts and become searchable in PubMed as well.
  • Are there ways to track a full list of our institutions OA articles in PMC (not picking up everything in PubMed)
    • Likely NIH will be contacting offices responsible for research to let them know what articles are out of compliance,  get their assistance in making sure that those get in.
    • Authors can easily update the electornic My Bibliography (in My NCBI in PubMed).
    • Author ID project, involves computational disambiguation. Where you are asked if you are the author of a paper if you didn’t include it. It may also be possible to have automatic reporting to the institutions.
  • What did it took politically to get the appropriation bill passed (PMC initiative)?
    • Congress always pushed more open access, because it was already spending money on the research. Most of the initiative came more from librarians (i.e. small libraries not having sufficient access) and government, than from the NIH.
  • Is there way to narrow down to NIH, free full text papers from PMC?
    • In PubMed, you can filter free full text articles in general via the limits.
  • Are all the articles deposited in PMC submitted the final manuscript?
    • Generally, yes.

HT: @bentoth on Twitter





Reclaim your Privacy on Facebook using a Simple Bookmarklet

20 05 2010

Of all social networking sites, Facebook causes the greatest privacy concerns. Certainly since it has changed its privacy options over time.

In the beginning, Facebook restricted the visibility of a user’s personal information to just their friends and their “network”, but the default privacy settings have become much more permissive, as you can see in the video below.
This short video is based on a visualization made by Matt McKeon and gives only an impression of a work-in-progress
(for up to date info check the original animation at http://mattmckeon.com/facebook-privacy/).

The reason? According Facebook founder Mark Zuckerberg the controversial new default and permanent settings just reflect the way the world has changed, becoming more public and less private (see ReadWriteWeb).

“Default” is the key to the problems. You have to opt out to protect your privacy. However to fully protect your privacy on Facebook, you have to navigate through 50 settings with more than 170 options (see great charts at the NY Times!). Facebook’s privacy policy is longer than the American constitution!!!

Shocked by the results of the ACLU’s Facebook Quiz (see Mashable), I already changed my privacy settings last summer. Doing a simple quiz on Facebook meant everything on your profile (whether you use privacy settings or not), is available to the quiz. Even more worrying, when your friends do a quiz, everything on your profile is made available to the developers as well.

Since the default privacy settings have changed, my settings needed to be adapted again. But where were the leaks in the 170 options?

Luckily there is a very simple bookmarklet Reclaim Privacy that can check and fix your profile in 2 minutes (see Mashable.com) It is very easy.

1. First go to Reclaim Privacy and drag the bookmarklet to your web browser bookmarks bar
(in the example I dragged the bookmarklet into Chrome’s bookmarks (upper arrow)

2. Go to your Facebook privacy settings and then click that bookmark (Scan for Privacy, see arrow) once you are on Facebook.

3. You will see a series of privacy scans that inspect your privacy settings and warn you about settings that might be unexpectedly public.
In my case my friends could still accidentally share my personal information. This is indicated by a red sign: “insecure.

4. So I clicked “prevent friends from sharing your data”, and in seconds this was the result:

5. I tweaked the contact information a bit (caution) by changing my contact settings, but I still would allow everyone to add me as a friend (I still have to approve, don’t I?)

Piece of cake!





When #Twitter Gets Creepy: People Who Force you to #Autofollow

18 05 2010

The third Twitter post in a row. But this one ain’t positive.

It is about privacy and spam.

Let’s first explain some basic things about Twitter.

People can follow you without your approval, at least if you  have a public account. You can follow them back if you like.

You just have to click on the follow button, that is all!
Everyone with a Twitter account can follow Barack Obama, for instance.

If Barack Obama followed me (whether I followed him or not), I could dm (direct message) him. He (or rather his staff) will receive a private message from me in his inbox.

Only people you follow, are able to dm you. This is to protect you against dm’s from whichever fool, Spam and Bots.

Barack Obama has many followers:  3,964,789. This is no surprise, because he is the president of the United States and everyone wants to know what he has to say.

Some people especially in the marketing sector find the numbers of followers that important that they will do anything to assure a lot of followers. They are even willing to pay for it.

There are several companies who specialize in it. Here is a list of paid Twitter services and their rates (from http://zacjohnson.com/buy-twitter-followers/).

  • BuyTwitterFriends.com = 10,000 Followers for $49.99 (0.0049 each)
  • TweetSourcer.com = 10,000 Followers for $60.00 (0.006 each)
  • UnlimitedTwitterFollowers.com = 10,000 Followers for $74.95 (0.0074 each)
  • Twitter1k.com = 5,000 Followers for $104.97 (0.0209 each)
  • SocialKik.com = 10,000 Followers for $150.00 (0.015 each)
  • USocial.net = 10,000 Followers for $447.30 (0.044 each)
  • Tweetcha.com = 10,000 Followers for $474.99 (0.047 each)
  • PurchaseTwitterFollowers.com = 5,000 Followers for $249.99 (0.049 each)

Buying followers….. that is rather shortsighted. My mother always used to say: “You can’t buy friends” (no real friends anyway).

What are followers worth who don’t follow you because you’re tweets are so interesting, or the stuff you sell is so good, etc… Do these followers really ‘follow’ you, in the sense that they follow what you say? And do they keep following you?

Moreover how are those followers recruited? Are they asked to do so? Are they offered money?

Well I don’t think so. There must be easier money strategies.

But how can you make people follow without asking?

Well there may be a sneaky way to force people to do so, without them being aware of it.

At least I think that has happened to me.

Saturday I got this dm:

I was alarmed.

  • First, somebody sends me a dm with a link to a marketing gift. I never click such links, you never know where they lead to. Even if it comes from someone I trust, it may be that his/her account has been hacked, so I have learned…
  • Second,  I’m not following this guy, at least not any longer….
  • Third I have blocked him before, after a similar dm.

The first time I got a dm of @jonathanvolk I thought I made the mistake by accidentally following him. But now (having blocked him before) I was sure that that wasn’t the case.

Out of curiosity (and to block him) I checked his Twitter account. Here I found several people complaining to him about the very same thing (the first tweet appeared later in response to my tweets).

@SorbetDigital appeared to have similar problems, not only with @jonathanvolk, but also with @JohnChow (see her post).

@JohnChow did ring a bell. Didn’t I block him in the past and didn’t I see his tweets rolling by lately?

I quickly checked Friend or Follow, a fantastic program, that shows you the people you follow and don’t follow you in return (following), your fans (who only follow you) and your friends (reciprocal relationship).

And who did I see there? John Chow, plus another guy that I presumably didn’t follow voluntarily: @MrGatherSuccess.
[The 2nd robot to the upper left also isn't kosher, as I found out today.]

Their Twitter pages ((below in blue and pink) have texts according to expectations.
Their follower/following ratio is absolutely skewed (557:1 and 1090:1 respectively)  so apparently their approach works in the sense that they got more followers, probably recruited in much the same way as they “recruited” me.

Strikingly@jonathanvolk and @Shoemoney are among the 100 people John Chow has chosen to follow. @Shoemoney (follower/-following ratio of 1355:1) and @Chow are also almost the only people followed by @MrGatherSuccess. By the way there is also “College Pages”, that links to Online Colleges, you know the site I warned you about on several occasions (Beware of Top 50 “Great Tools to Double Check your Doctor” or whatever Lists and “Vanity is the Quicksand of Reasoning: Beware of Top 100 and 50 lists!”)

Oh and “the robot” tweeted this today

Common features of these people:

  • they are  all Internet marketers,
  • All have let me follow them, (without following me back)
  • Some have sent me dm’s
  • they have many followers, some having skewed follower/following ratios
  • they “know” each other and may refer to each other

Strikingly @jonathanvolk has a post in which he explains how to get 25,000 Twitter followers with “Twitter Followers for Sale”. Juicy detail: Shoemoney gave him the tip. Vice versa at shoemoney.com, Shoemoney advocates to download the affiliate marketing guide of Jonathan.

Are these the guys behind the link services?

Not necessarily. In a recent post (Something Fishy Goin’ On Here… Paid Twitter Followers) @Jonathanvolk seems sincerely surprised about the pissed of reaction of his forced followers. Quote:

The other week I made a post about Paid twitter followers.

In the post I outline a few methods I have used to essentially pay for twitter followers and how much it has cost me per follower. With the methods like paying twittercounter.com, for example, you know exactly where your twitter followers are coming from.

Recently my follower count has been increasing steadily (and fairly rapidly) without me paying for any more services.

I’ve received a few @ messages before saying the person didn’t follow me and they were unsure how they did. I usually brushed it off as a… how can I put it lightly… computer illiterate person.(emphasis mine)

I think however that one of the services I used is using some sort of application access to automatically make users follow those who pay for the service.

The only problem is, I’m not sure which service is doing it… or if it’s just someone trying to get my account banned.

Since I have no way of know knowing… I have no way of stopping it.

Kinda crazy. Either way, be careful buying followers unless you know explicitly where the users are coming from!

——————

Kinda bullshitSince I have no way of know knowing… I have no way of stopping it.” …. Booh!

Let me give you one tip, guys (assuming that you are honest about this): go sit around the table and see which follower-robbering service you share, and do something about it!!

How people can force you to follow is a technical issue, I know little about. Jonathan refers to a follow bug in Twitter that they have found but should have been fixed.

Indeed @librarianbe told me the same in response to my “tweets for help”. He referred to an article in Gizmodo explaining how to force anyone to follow you on Twitter. Apparently the bug was not fixed (yet?), or there is another leak still to be discovered.

Twitter handled the p @  r  n-spam well. I hope it will find a solution to these problems too.

For such forced following and marketing dm’s are not only annoying, and an intrusion on our privacy, they are also bad for the credibility of a tool like Twitter.

So I’m going to block these guys (of course) and report them to Twitter using the ticket file @mrgunn advised me.

Similar problems? Here is the link to file a ticket with Twitter: http://help.twitter.com/requests/new

Meanwhile I advise you marketer guys to reassess the value of your followers. Do you only care about the size of the flock? Is it just the number of sheep? Do you want to impress by numbers? Or do you care about by whom you are being followed? And if what you’re tweeting does matter to them? Because only then you will have value as a twitterer and deserve to be followed. Otherwise, how can I put it lightly…you’re  a bit sheepish.

Added 18-05-2010

According to Twitter Status the bug that permitted a user to “force” other users to follow them was resolved & cleaned up May 10th. However Jonathan send the dm May 15th (although he might have forced me to follow him longer ago).

If you are still seeing folks you are following who you didn’t choose to follow, Twitter advises to use the block or unfollow tools as a remedy.

However, these buttons do not work effectively as @jonathanvolk and @johnchow keep resurrecting again after a total block.

@jonathanvolk reappeared in the Following Tab of Friend or Follow this very night, about 3 days after blocking (see comment).

Twitter, I hope you listen…





A Quantitave Study suggests that Twitter is not Primarily a Social Networking Site

13 05 2010

A lot can be said about Twitter, Facebook, Linkedin and other social media. What is the best, the most useful, the most popular the most social (and has the least privacy-issues, hehe Facebook)?

You know I love Twitter. Twitter is a social networking and microblogging service that enables its users to send and read messages known as tweets. The tweets don’t exceed 140 characters, so your message must be very concise. For me Twitter is a very rich source of information and a useful networking site. But it is hard to explain that to others.

Some Most people think that individuals who twitter are just parroting others (hé this is called retweeting, guys!) or are just egocentric bores (“I eat cornflakes for dinner”).

Well, a recent quantitative study by a group of researchers at Korea’s Advanced Institute of Science and Technology suggest that they might just be right. … Or at least their data suggest Twitter may be less of a social site and more of a news site.

According to Haewoon Kwak et al this is the first quantitative Twitter study ever.

The researchers crawled the entire Twitter site and obtained 41.7 million user profiles, 1.47 billion social relations, 4,262 trending topics, and 106 million tweets. They looked at the follower-following topology, looked at the ranking by number of followers and by PageRank, analyzed the retweets and the tweets of top trending topics.

You can read the main conclusions in the power presentation below and their abstract for Proceedings of the 19th International World Wide Web (WWW) Conference, April 26-30, 2010, Raleigh NC (USA). Below the abstract you can also find links to two download files, enabling you to reanalyze the data
Going Social Now and ReadWriteWeb also give a nice overview.

What are their main conclusions:

  • Twitter is not very “social”
    • It is “I follow you”, not “lets become friends” and you don’t have to approve or follow back. Following thus means that you “just subscribe” to the tweets of that person.
    • Only 22.1% of the relationships are reciprocal, thus 77.9% of the relationships is one way, just one of two is following the other. Surprisingly, 67.6% of users on Twitter are not followed by any of the people they follow.
    • this low reciprocity is unlike all other human social networks.
  • For most tweople, Twitter is primarily a source of information, not a social networking or information dissemination platform.
    • The Majority of topics (54,3%) are headline topics
    • Few users reach a large audience directly.
    • The average path length between two people on Twitter is 4.12. This is much shorter than Stanley Milgram’s original experiment uncovering the “six degrees of separation” phenomena.
    • Any retweeted tweet is to reach an average of 1,000 users no matter what the number of followers is of the original tweet.
    • Once retweeted, a tweet gets retweeted almost instantly on next hops, signifying fast diffusion of information after the 1st retweet.

It is a beautiful study that highlights the topological characteristics of Twitter.

One word of caution. Twitter is analyzed as a whole. There are many subpopulations with their own kinetics and goals. So the majority of people may follow the news, and fans may follow a celebrity by the million, but there are (relatively) small niches on Twitter, like health and medicine (or science) that may not follow the same rules.
I daresay (guess) that more people in this niche follow each other and do use Twitter both as a source of information and as as  network for social communication.
But these small niches are outnumbered by others (news sites, CEOs, celebrities).
At least that is my hypothesis.

Who is going to test this??

Many different Twitter birds in a flock

Credits





Three Studies Now Refute the Presence of XMRV in Chronic Fatigue Syndrome (CFS)

27 04 2010

ResearchBlogging.org.“Removing the doubt is part of the cure” (RedLabs)

Two months ago I wrote about two contradictory studies on the presence of the novel XMRV retrovirus in blood of patients with Chronic Fatigue Syndrome (CFS).

The first study, published in autumn last year by investigators of the Whittemore Peterson Institute (WPI) in the USA [1], claimed to find XMRV virus in peripheral blood mononuclear cells (PBMC) of patients with CFS. They used PCR and several other techniques.

A second study, performed in the UK [2] failed to show any XMRV-virus in peripheral blood of CFS patients.

Now there are two other negative studies, one from the UK [3] and one from the Netherlands [4].

Does this mean that XMRV is NOT present in CFS patients?

No, different results may still be due do to different experimental conditions and patient characteristics.

The discrepancies between the studies discussed in the previous post remain, but there are new insights, that I would like to share.*

1. Conflict of Interest, bias

Most CFS patients seem “to go for” WPI, because WPI, established by the family of a chronic fatigue patient, has a commitment to CFS. CFS patients feel that many psychiatrists, including authors of the negative papers [2-4] dismiss CFS as something “between the ears”.  This explains the negative attitude against these “psych-healers” on ME-forums (i.e. the Belgium forum MECVS.net and http://www.forums.aboutmecfs.org/). MECVS even has a section “faulty/wrong” papers, i.e. about the “failure” of psychiatrists to demonstrate  XMRV!

Since a viral (biological) cause would not fit in the philosophy of these psychiatrists, they might just not do their best to find the virus. Or even worse…

Dr. Mikovits, co-author of the first paper [1] and Director of Research at WPI, even responded to the first UK study as follows (ERV and Prohealth):

“You can’t claim to replicate a study if you don’t do a single thing that we did in our study,” …
“They skewed their experimental design in order to not find XMRV in the blood.” (emphasis mine)

Mikovits also suggested that insurance companies in the UK are behind attempts to sully their findings (ERV).

These kind of personal attacks are “not done” in Science. And certainly not via this route.

Furthermore, WPI has its own bias.

For one thing WPI is dependent on CFS and other neuro-immune patients for its existence.

WPI has generated numerous press releases and doesn’t seem to use the normal scientific channels. Mikovits presented a 1 hr Q&A session about XMRV and CFS (in a stage where nothing has been proven yet). She will also present data about XMRV at an autism meeting. There is a lot of PR going on.

Furthermore there is an intimate link  between WPI and VIP Dx, both housed in Reno. Vip DX is licensed by WPI to provide the XMRV-test. Vipdx.com links to the same site as redlabsusa.com, for Vip Dx is the new name of the former RedLabs.

Interestingly Lombardi (the first author of the paper) co-founded Redlabs USA Inc. and  served as the Director of Operations at Redlabs, Harvey Whittemore owns 100% of VIP Dx, and was the company President until this year and  Mikovits is the Vice President of VIP Dx. (ME-forum). They didn’t disclose this in the Science paper.

TEST_RQN_Feb_2010

Vip/Dx offers a plethora of tests, and is the only RedLab -branch that performs the WPI-PCR test, now replaced by the “sensitive” culture test (see below). At this stage of controversy, the test is sold as “a reliable diagnostic tool“(according to prohealth). Surely their motto “Removing the doubt is part of the cure” appeals to patients. But how can doubt be removed if the association of XMRV with CFS has not been confirmed, the diagnostic tests offered have yet not been truly validated (see below), as long as a causal relationship between XMRV and CFS has not been proven and/or when XMRV does not seem that specific for CFS: it has also been found in people with prostate cancer, autism,  atypical multiple sclerosis, fibromyalgia, lymphoma)(WSJ).

Meanwhile CFS/ME websites are abuzz with queries about how to obtain tests -also in Europe- …and antiretroviral drugs. Sites like Prohealth seem to advocate for WPI. There is even a commercial XMRV site (who runs it is unclear)

Project leader Mikovits, and the WPI as a whole, seem to have many contacts with CSF patients, also by mail. In one such mail she says (emphasis and [exclamations] mine):

“First of all the current diagnostic testing will define with essentially 100% accuracy! XMRV infected patients”. [Bligh me!]….
We are testing the hypothesis that XMRV is to CFS as HIV is to AIDS. There are many people with HIV who don’t have AIDS (because they are getting treatment). But by definition if you have ME you must have XMRV. [doh?]
[....] There is so much that we don’t know about the virus. Recall that the first isolation of HIV was from a single AIDS patient published in late 1982 and it was not until 2 years later that it was associated with AIDS with the kind of evidence that we put into that first paper. Only a few short years later there were effective therapies. [...]. Please don’t hesitate to email me directly if you or anyone in the group has questions/concerns. To be clear..I do think even if you tested negative now that you are likely still infected with XMRV or its closest cousin..

Kind regards, Judy

These tests costs patients money, because even Medicare will only reimburse 15% of the PCR-test till now. VIP Dx does donate anything above costs to XMRV research, but isn’t this an indirect way to support the WPI-research? Why do patients have to pay for tests that have not proven to be diagnostic? The test is only in the experimental phase.

I ask you: would such an attitude be tolerated from a regular pharmaceutical company?

Patients

Another discrepancy between the WPI and the other studies is that only the WPI use the Fukuda and Canadian criteria to diagnose CFS patients. The Canadian  criteria are much more rigid than those used in the European studies. This could explain why WPI has more positives than the other studies, but it can’t fully explain that WPI shows 96% positives (their recent claim) against 0% in the other studies. For at least some of the European patients should fulfill the more rigid criteria.

Regional Differences

Patients of the positive and negative studies also differ with respect to the region they come from (US and Europe). Indeed, XMRV has previously been detected in prostate cancer cells from American patients, but not from German and Irish patients.

However, the latter two reasons may not be crucial if the statement in the open letter* from Annette Whittemore, director of the WPI, to Dr McClure**, the virologist of the second paper [2], is true:

We would also like to report that WPI researchers have previously detected XMRV in patient samples from both Dr. Kerr’s and Dr. van Kuppeveld’s cohorts prior to the completion of their own studies, as they requested. We have email communication that confirms both doctors were aware of these findings before publishing their negative papers.(……)
One might begin to suspect that the discrepancy between our findings of XMRV in our patient population and patients outside of the United States, from several separate laboratories, are in part due to technical aspects of the testing procedures.

Assuming that this is true we will now concentrate on the differences in the PCR -procedures and results.

PCR

All publications have used PCR to test the presence of XMRV in blood: XMRV is present in such low amounts that you can’t detect the RNA without amplifying it first.

PCR allows the detection of a single or few copies of target DNA/RNA per milligram DNA input, theoretically 1 target DNA copy in 105 to 106 cells. (RNA is first reverse transcribed to DNA). If the target is not frequent, the amplified DNA is only visible after Southern blotting (a radioactive probe “with a perfect fit to” the amplified sequence) or after a second PCR round (so called nested PCR). In this second round a set of primers is used internal to the first set of primers. So a weak signal is converted in a strong and visible one.

All groups have applied nested PCR. The last two studies have also used a sensitive real time PCR, which is more of a quantitative assay and less prone to contamination.

Twenty years ago, I had similar experiences as the WPI. I saw very vague PCR bands that had all characteristics of a tumor-specific sequence in  normal individuals, which was contrary to prevailing beliefs and hard to prove. This had all to do with a target frequency near to the detection limit and with the high chance of contamination with positive controls. I had to enrich tonsils and purified B cells to get a signal and sequence the found PCR products to prove we had no contamination. Data were soon confirmed by others. By the way our finding of a tumor specific sequence in normal individuals didn’t mean that everyone develops lymphoma (oh analogy)

Now if you want to proof you’re right when you discovered something new you better do it good.

Whether a PCR assay at or near the detection limit of PCR is successful depends on:

  • the sensitivity of the PCR
    • Every scientific paper should show the detection limit of the PCR: what can the PCR detect? Is 1 virus particle enough or need there be 100 copies of the virus before it is detected? Preferably the positive control should be diluted in negative cells. This is called spiking. Testing a positive control diluted in water doesn’t reflect the true sensitivity. It is much easier for primers to find one single small piece of target DNA in water than to find that piece of DNA swimming in a pool of DNA from 105 cells. 
  • the specificity of the PCR.
    • You can get aspecific bands if the primers recognize other than the intended sequences. Suppose you have one target sequence competing with a lot of similar sequences, then even a less perfect match in the normal genome has every chance to get amplified. Therefore you should have a negative control of cells not containing the virus (i.e. placental DNA), not only water. This resembles the PCR conditions of your test samples.
  • Contamination
    • this should be prevented by rigorous spatial separation of  sample preparation, PCR reaction assembly, PCR execution, and post-PCR analysis. There should be many negative controls. Control samples should be processed the same way as the experimental samples and should preferably be handled blinded.
  • The quality and properties of your sample.
    • If XMRV is mainly present in PBMC, separation of PBMC by Ficoll separation (from other cells and serum) could make the difference between a positive and a negative signal. Furthermore,  whole blood and other body fluids often contain inhibitors, that may lead to a much lower sensitivity. Purification steps are recommended and presence of inhibitors should be checked by spiking and amplification of control sequences.

Below the results per article. I have also made an overview of the results in a Google spreadsheet.

WPI
The PCR conditions are badly reported in the WPI paper, published in Science[1]. As a matter of fact I wonder how it ever came trough the review.

  • Unlike XMRV-positive prostate cancer cells, XMRV infection status did not not correlate with the RNASEL genotype.
  • The sensitivity of the PCR is not shown (nor discussed).
  • No positive control is mentioned. The negative controls were just vials without added DNA.
  • Although the PCR is near the detection limit, only first round products are shown (without confirmation of the identity of the product). The positive bands are really strong, whereas you expect them to be weak (near the detection limit after two rounds). This is suggestive of contamination.
  • PBMC have been used as a source and that is fine, but one of WPI’s open letters/news items (Feb 18), in response to the first UK study, says the following:
    • point 7. Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells.(emphasis mine)
  • But carefully reading the methods,  mentioned in the “supporting material” I only read:
    • The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 ºC for further culture and analysis or resuspended in TRIzol (…) and stored at -80 ºC for DNA and RNA extraction and analysis. (emphasis mine)

    Either …. or. Seems clear to me that the PBMC were not cultured for PCR, at least not in the experiments described in the science paper.

    How can one accuse other scientists of not “duplicating” the results if the methods are so poorly described and the authors don’t adhere to it themselves??

  • Strikingly only those PCR-reactions are shown, performed by the Cleveland Clinic (using one round), not the actual PCR-data performed by WPI. That is really odd.
  • It is also not clear whether the results obtained by the various tests were consistent.
    Suzanne D. Vernon, PhD, Scientific Director of the CFIDS Association of America (charitable organization dedicated to CFS) has digged deeper into the topic. This is what she wrote [9]:
    Of the 101 CFS subjects reported in the paper, results for the various assays are shown for only 32 CFS subjects. Of the 32 CFS subjects whose results for any of the tests are displayed, 12 CFS subjects were positive for XMRV on more than one assay. The other 20 CFS subjects were documented as positive by just one testing method. Using information from a public presentation at the federal CFS Advisory Committee, four of the 12 CFS subjects (WPI 1118, 1150, 1199 and 1125) included in the Science paper were also reported to have cancer – either lymphoma, mantle cell lymphoma or myelodysplasia. The presentation reported that 17 WPI repository CFS subjects with cancer had tested positive for XMRV. So how well are these CFS cases characterized, really?

The Erlwein study was published within 3 months after the first article. It is simpler in design and was reviewed in less then 3 days. They used whole blood instead of PBMC and performed nested PCR using another set of primers. This doesn’t matter a lot, if the PCR is sensitive. However, the sensitivity of the assay is not shown and the PCR bands of the positive control look very weak, even after the second round (think they mad a mistake in the legend as well: lane 9 is not a positive control but a base pair ladder, I presume). It also looked like they used a “molecular plasmid control in water”, but in the comments on the PLoS ONE paper, one of the authors states that the positive control WAS spiked into patient DNA.(Qetzel commenting to Pipeline Corante) Using this PCR none of the 186 CSF samples was positive.

Groom and van Kuppeveld studies
The two other studies use an excellent PCR approach[3,4]. Both used PBMC, van Kuppeveld used older cryoperserved PBMC. They first tried the primers of Lombardi using a similar nested PCR, but since the sensitivity was low they changed to a real time PCR with other optimized primers. They determined the sensitivity of the PCR by serially diluting a plasmid into PBMC DNA from a healthy donor. The limit of sensitivity equates to 16 and 10 XMRV-gene copies in the UK and the Dutch study respectively. They have appropriate negative controls and controls for the integrity of the material (GAPDH, spiking normal control cDNAs in negative DNA to exclude sample mediated PCR inhibition[1], phocine distemper virus[2]), therefore also excluding that cryopreserved PBMC were not suitable for amplification.

The results look excellent, but none of the PCR-samples were positive using these sensitive techniques. A limitation of the Dutch study is the that numbers of patients and controls were small (32 CSF, 43 controls)

Summary and Conclusion

In a recent publication in Science, Lombardi and co-authors from the WPI reported the detection of XMRV-related, a novel retrovirus that was first identified in prostate cancer samples.

Their main finding, presence of XMRV in peripheral blood cells could not be replicated by 3 other studies, even under sensitive PCR conditions.

The original Science study has severe flaws, discussed above. For one thing WPI doesn’t seem to adhere to the PCR to test XMRV any longer.

It is still possible that XMRV is present in amounts at or near the detection limit. But it is equally possible that the finding is an artifact (the paper being so inaccurate and incomplete). And even if XMRV was reproducible present in CFS patients, causality is still not proven and it is way too far to offer patients “diagnostic tests” and retroviral treatment.

Perhaps the most worrisome part of it all is the non-scientific attitude of WPI-employees towards colleague-scientists, their continuous communication via press releases. And the way they try to directly reach patients, who -i can’t blame them-, are fed up with people not taking them serious and who are longing for a better diagnosis and most of all a better treatment. But this is not the way.

Credits

*Many thanks to Tate (CSF-patient) for alerting me to the last Dutch publication, Q&A’s of WPI and the findings of Mrs Vernon.
- Ficoll blood separation. Photo [CC] http://www.flickr.com/photos/42299655@N00/3013136882/
– Nested PCR: ivpresearch.org

References

  1. Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, & Mikovits JA (2009). Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science (New York, N.Y.), 326 (5952), 585-9 PMID: 19815723
  2. Erlwein, O., Kaye, S., McClure, M., Weber, J., Wills, G., Collier, D., Wessely, S., & Cleare, A. (2010). Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008519
  3. Groom, H., Boucherit, V., Makinson, K., Randal, E., Baptista, S., Hagan, S., Gow, J., Mattes, F., Breuer, J., Kerr, J., Stoye, J., & Bishop, K. (2010). Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome Retrovirology, 7 (1) DOI: 10.1186/1742-4690-7-10
  4. van Kuppeveld, F., Jong, A., Lanke, K., Verhaegh, G., Melchers, W., Swanink, C., Bleijenberg, G., Netea, M., Galama, J., & van der Meer, J. (2010). Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort BMJ, 340 (feb25 1) DOI: 10.1136/bmj.c1018
  5. McClure, M., & Wessely, S. (2010). Chronic fatigue syndrome and human retrovirus XMRV BMJ, 340 (feb25 1) DOI: 10.1136/bmj.c1099
  6. http://scienceblogs.com/erv/2010/01/xmrv_and_chronic_fatigue_syndr_5.php
  7. http://scienceblogs.com/erv/2010/01/xmrv_and_chronic_fatigue_syndr_6.php
  8. http://scienceblogs.com/erv/2010/03/xmrv_and_chronic_fatigue_syndr_11.php
  9. http://www.cfids.org/xmrv/022510study.asp
Sensitivity of PCR screening for XMRV in PBMC DNA. VP62 plasmid was serially diluted 1:10 into PBMC DNA from a healthy donor and tested by Taqman PCR with env 6173 primers and probe. The final amount of VP62 DNA in the reaction was A, 2.3 × 10-2 ng, B, 2.3 × 10-3 ng, C, 2.3 × 10-4 ng, D, 2.3 × 10-5 ng, E, 2.3 × 10-6 ng, F, 2.3 × 10-7 ng or G, 2.3 × 10-8 ng. The limit of sensitivity was 2.3 × 10-7 ng (shown by trace F) which equates to 16 molecules of VP62 XMRV clone.




Silly Saturday #22 – A Picture is Worth a 1000 Words.

17 04 2010

This post is my submission for the Grand Rounds to be hosted at Sterile Eye.
This upcoming edition has the theme VISUAL COMMUNICATION.

You know I love visualizations, they are so easy to understand.

No lengthy post here, because a picture is worth a 1000 words…..

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I

250lbs versus 120 lbs

The body scans side by side of 250 lb. woman versus 120 lb. woman.
Source: Bored Panda

Hattip: @EvidenceMatters, @rlbates & @streetanatomy who referred to a repost on LikeCool

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II

Planes or Volcano?

We were wondering this today (April 16, 2010)

Source: Information is Beautiful

Hattip: Bitethedust & @mpesce “Turns out that a little volcanic action is surprisingly good for planet Earth They referred to a repost at The Daily Wh.at.

I’m a real fan of Information is Beautiful with its beautiful visualizations. See previous post on evidence for health supplements

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III

Real Eyeballing

Source: Wolfram Demonstrations Project

Contributed to Sterile Eye ;) : An interactive project showing hows the interaction between an eyeball and two of the muscles connected to it. Muscles deform as the eyeball rotates. You can download a live version.





Irreversible Effects of Previous Cortisol Excess on Cognitive Functions in Cushing’s Disease

10 04 2010

ResearchBlogging.orgApril 8th is Cushing’s Awareness Day. This day has been chosen as a day of awareness as it is the birthday of Dr. Harvey Cushing, a neurosurgeon, who discovered this illness.

Cushing’s disease is a rare hormone disease caused by prolonged exposure to high levels of the stress hormone cortisol in the blood, whereas Addison’s disease is caused by the opposite: the lack of cortisol. For more background information on both see this previous post. Ramona Bates MD, of Suture for a Living, has written an excellent review (in plain language) about Cushing’s Disease on occasion of Cushing Awareness Day at EmaxHealth.

From this you can learn that Cushing’s disease can be due to the patient taking cortisol-like glucocorticoids, such as prednisone for asthma (exogenous cause), but can also arise because people’s bodies make too much of cortisol itself.  This may be due to a tumor on the pituitary gland, the adrenal gland, or elsewhere in the body.

Symptoms of Cushing’s disease are related to the effects of high levels of cortisol or other glucocorticoids on the immune system, the metabolism and  the brain. Symptoms include rapid weight gain, particularly of the trunk and face (central obesity, “moon face” and buffalo neck), thinning of the skin and easy bruising, excessive hair growth, opportunistic infections, osteoporosis and high blood pressure.

Less emphasized than the clinical features are the often very disabling cognitive deficits and emotional symptoms that accompany Cushing’s disease. Cushing patients may suffer from various psychological disturbances, like insomnia, mood swings, depression and manic depression, and from cognitive decline. Several studies have shown that these glucocorticoid induced changes are accompanied by atrophy of the brain, and in particular of the  hippocampal region, leading to hippocampal volume loss and a profound loss of synapses [2]. This hippocampal loss seems reversible [2], but are neurological and psychological defects also restored? This is far more important to the patient than anatomic changes.

If we listen to Cushing patients, who are “cured” and have traded Cushing’s disease for Addison’s disease, we notice that they feel better after their high levels of cortisol have normalized, but not fully cured (see two examples of ex-Cushing patients with longlasting if not irreversible health) problems in my previous post here. [added 2010-04-17)
To realize how this affects daily life, I recommend to read the photo-blog 365 days with Cushing by Robin (also author of Survive the Journey). Quite a few of her posts deal with the continuous weakness (tag muscle atrophy), tiredness (tag fatigue), problems with (short-term) memory (see tag memory)  or both (like here and here).

Scientifically the question is to which extent ex-Cushing patients score worse than other healthy individuals or chronically ill people and, if so, whether this can be attributed to the previous high levels of glucocorticoids.

A recent study by endocrinologists (and one neurologists) from the Leiden University Medical Center assessed the cognitive functioning of patients  after long-term cure of their Cushing’s disease (caused by a ACTH producing pituitary adenoma, that induces overproduction of cortisol (hypercortisolism) by the adrenals [1]. Previous studies had contradictory outcomes and/or were too small to draw conclusions.

The authors first compared a group of 74 Cushing patients (with a previous pituitary tumor) with matched healthy controls (selected by the patients themselves). Matched means that these controls had the same characteristics as the Cushing patients with respect to gender (male/female: 13/61), age (52 yr) and education.
Cushing patients were on average 13 years in remission and were followed for another 3 years (total 16 yrs follow-up). Cushing’s disease  had been established by clinical signs and symptoms and by appropriate biochemical tests. All patients were treated by transsphenoidal surgery (surgery via the nostrils), if necessary followed by repeat surgery and/or radiotherapy (27%). Cure of Cushing’s disease was defined by normal overnight suppression of plasma cortisol levels after administration of dexamethasone and normal 24-h urinary excretion rates of cortisol. 58% of the patients had at least one form of hypopituitarism (deficiency of one or more hormones) and half of the patients needed hydrocortisone replacement therapy.

Long after their cure, 62% of the Cushing patients reported memory problems, and 47% reported problems in executive functioning. The Hospital Anxiety and Depression Scale (HADS)-score (10.5)  indicated no clinical depression or anxiety. Patients with long-term cure of Cushing’s disease did not perform worse on measures of global cognitive functioning. However, these patients had several other cognitive impairments, mainly in the memory domain.
Only a single test result (FAS, measures verbal mental flexibility and fluency) was significantly different between patients with short and long-term remission.

From direct comparison with healthy controls it is not clear what causes these cognitive alterations in Cushing patients.

Therefore the cognitive function of Cushing patients was compared to that of patients previously treated for non-functioning pituitary macroadenomas (NFMA).
NFMA patients were chosen, because they have undergone similar treatments (transsphenoidal surgery (100%), with repeat surgery and/or radiotherapy (44%) as the Cushing patients. They also shared hypopituitarism and the need for hydrocortisone substitution in half of the cases. NFMA patients, however, have never been exposed to prolonged excess of cortisol.

Cushing patients could not be directly compared to NFMA-patients, because these patient groups differed with regard to age and gender.

Thus Cushing patients were compared to matched healthy controls and NFMA to another set of healthy controls, matched to these NFMA patients (Male/Female: 30/24  and mean age: 61 yr).

To compare Cushing patients with NFMA patients the Z-scores* were calculated for each patient group in relation to their appropriate control group. A general linear model was used to compare the Z-scores.

Overall Cushing patients performed worse than NFMA patients. In the memory domain, patients cured from Cushing’s disease had a significantly lower MQ measured with the Wechsler Memory Scale compared with patients with NFMA in the subscales concentration and visual memory. On the Verbal Learning Test of Rey, patients cured from Cushing’s disease recalled fewer words in the imprinting, the immediate and delayed recall trials. Furthermore, on the Rey Complex Figure, patients with cured Cushing’s disease scored worse on both trials when compared with NFMA patients. In tests measuring executive function, patients cured from Cushing’s disease made fewer correct substitutions on the Letter-Digit Substitution Test and came up with fewer correct patterns on the Figure Fluency Test compared with treated NFMA patients.

These impairments were not merely related to pituitary disease in general and/or its treatment, because these patients with long-term cure of Cushing’s disease also revealed subtle impairments in cognitive function compared with patients previously treated for NFMA. These are most likely caused by the irreversible effects of previous glucocorticoid excess on the central nervous system (because this is the main difference between the two).

Sub-analysis indicated that hypopituitarism was associated with mildly impaired executive functioning** and hydrocortisone dependency** and additional radiotherapy were negatively associated with memory and executive functioning, whereas the duration of remission positively influenced memory and executive functioning.

The main point of criticism, apparently raised during the review process and discussed by the authors, is the presentation of the data without adjustments for multiple comparisons. When more than one test is used, the chance of finding at least one test statistically significant due to chance increases. As the authors point out, however, the positive significant results were not randomly distributed among the different variables. Furthermore, the findings are plausible given the irreversible effects of cortisol excess on the central nervous system in experimental animal and clinical studies.

Although not addressed in this study, similar cognitive impairments would be expected in patients having continuous overexposure to exogenous glucocorticosteroids, like prednison.

* Z-scores: The z score for an item, indicates how far and in what direction, that item deviates from its distribution’s mean, expressed in units of its distribution’s standard deviation. The z score transformation is especially useful when seeking to compare the relative standings of items from distributions with different means and/or different standard deviations (see: http://sysurvey.com/tips/statistics/zscore.htm).

** This makes me wonder whether Addison patients with panhypopituitarism have lower cognitive functions compared to healthy controls as well.

Hattip: Hersenschade door stresshormoon lijkt onomkeerbaar (2010/04/08/) (medicalfacts.nl/)

References

  1. Tiemensma J, Kokshoorn NE, Biermasz NR, Keijser BJ, Wassenaar MJ, Middelkoop HA, Pereira AM, & Romijn JA (2010). Subtle Cognitive Impairments in Patients with Long-Term Cure of Cushing’s Disease. The Journal of clinical endocrinology and metabolism PMID: 20371667
  2. Patil CG, Lad SP, Katznelson L, & Laws ER Jr (2007). Brain atrophy and cognitive deficits in Cushing’s disease. Neurosurgical focus, 23 (3) PMID: 17961025 Freely available PDF, also published at Medscape







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