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Published: November 2009Print Record of Viewing
Dr. Binnicker provides an overview of the new interferon gamma release assays to aid in the detection of tuberculosis infection. He also discusses the global impact of tuberculosis(TB), historic perspective on diagnosis, and the recent development of an in vitro whole blood assay for use in the detection of latent TB infection and active TB disease.
Presenter: Matt Binnicker, PhD, D(ABMM), Assistant Professor of Laboratory Medicine and Pathology, Director of the Infectious Diseases Serology Laboratory and Associate Director of the Mycology/Mycobacteriology Laboratory in the Division of Clinical Microbiology at Mayo Clinic
Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.
Our presenter for this program is Dr. Matthew Binnicker, Assistant Professor of Laboratory Medicine and Pathology, Director of the Infectious Diseases Serology Laboratory and Associate Director of the Mycology/Mycobacteriology Laboratory in the Division of Clinical Microbiology at Mayo Clinic. Dr. Binnicker will provide an overview of the new interferon gamma release assays to aid in the detection of tuberculosis infection. He will also discuss the global impact of tuberculosis (TB), historic perspective on diagnosis, and the recent development of an in vitro whole blood assay for use in the detection of latent TB infection and active TB disease.
In this presentation, we’ll review an exciting and relatively new technology, known as interferon gamma release assay, which is now available to assist in the diagnosis of tuberculosis. First, we’ll discuss the overall principle of this technology, and then I’ll turn our focus to a specific interferon gamma release assay, QuantiFERON TB Gold In-Tube, which we’ve recently implemented into the Infectious Disease serology laboratory at Mayo Clinic. I’ll review the potential applications of this method, its advantages and limitations in comparison to conventional techniques, and then we’ll go over some keys to accurate test performance that may be helpful to you as your hospital or laboratory is considering using this method.
Although there are significant advantages of QuantiFERON over conventional testing, there are also several limitations to this method. Despite QuantiFERON showing enhanced specificity, studies have shown that this method may still demonstrate cross-reactivity with some nontuberculous mycobacterial infections, including those caused by Mycobacterium kansasii, Mycobacterium szulgai and Mycobacterium marinum. A second limitation is the paucity of data for QuantiFERON in certain patient populations, including young children, immunocompromised patients, and pregnant women. Due to the lack of studies with these groups of patients, the interpretation of QuantiFERON results can be difficult, and future studies are needed.
Now I’d like to turn our attention to the applications of QuantiFERON and also discuss the assay’s key performance characteristics. I think one of the major questions that clinicians and laboratorians are asking is “Can interferon gamma release assays, such as QuantiFERON, be used in place of the tuberculin skin test?” In 2005, a panel of experts in the field of TB diagnostics, including members of the CDC, drafted a guidance document on the use and interpretation of QuantiFERON. In this document, the group concluded that QuantiFERON can be used in all circumstances in which the tuberculin skin test is used, including the following scenarios; contact investigations, evaluation of recent immigrants with a history of BCG vaccination, TB screening of health care workers, and serial evaluation for the diagnosis of TB infection.
So given these recommendations, I think another common question is “Should interferon gamma release assays always be used in place of the tuberculin skin test?” Unfortunately, I think the jury is still out on this, and more data is needed before we can definitively address this question. However, several national health organizations have issued potential algorithms for the use of assays such as QuantiFERON. In 2006, the UK’s National Institute for Health and Clinical Excellence issued TB diagnostic guidelines that provided a recommendation on the use of interferon gamma release assays. This group recommended a 2 step approach, in which patients are first tested by the skin test, and if positive, an interferon gamma release assay is performed. If the positive skin test is not confirmed by the interferon gamma release assay, the positive skin test should be considered a false positive result.
While this has been an algorithm that has been adopted by certain groups, I believe there are situations where initial testing by QuantiFERON may be useful. These situations would first include testing patients with a known history of BCG vaccination. Testing this group by QuantiFERON may significantly reduce the number of false positive skin test results, and subsequently reduce the costs associated with follow-up visits and diagnostic testing. A second scenario where primary testing by QuantiFERON makes sense is in health care workers and annual hospital employee screening. The requirement for a single visit for these individuals would reduce the time and costs associated with being away from work for follow-up visits.
What I’d like to do now is review the findings of a recent study that provide data to support the use of interferon gamma release assays in the situations we’ve discussed. This study published in 2008 by Pai et al was a systematic review, in which the authors performed a meta-analysis of 38 studies comparing interferon gamma release assays to the tuberculin skin test. The sensitivity was assessed using microbiologically confirmed TB cases, and the specificity was measured using healthy, low-risk individuals without known exposure to TB.
This meta-analysis found that the pooled sensitivity of the tuberculin skin test was 77%, versus 70% by QuantiFERON TB Gold In-Tube. This difference did not appear to reach statistical significance due to the overlapping 95% confidence intervals.
It does raise the question, “Does QuantiFERON In-Tube demonstrate a lower sensitivity compared to the tuberculin skin test?”
This question is challenging to answer, because it’s difficult to accurately estimate the sensitivity of these assays due to the absence of a gold standard method to arbitrate discordant results.
However, the CDC has stated that “A greater rate of positive results with the skin test than with QuantiFERON has been observed in persons WITH and WITHOUT recognized risks of Mycobacterium tuberculosis infection.” So I think this issue remains somewhat controversial.
On the next slide, we see a summary of the specificity data from this study, and this is where the major difference was observed between the skin test and QuantiFERON In-Tube. If we first look at the specificity data for those patients that had a history of BCG vaccination, the tuberculin skin test demonstrated a pooled specificity of 59%, while QuantiFERON In-Tube showed a significantly higher specificity of 96%.
This significant difference was not observed in patients WITHOUT a history of BCG vaccination, where the skin test and QuantiFERON In-Tube demonstrated pooled specificities of 97% and 99%, respectively.
These data suggest that the skin test and QuantiFERON appear to have similar sensitivity for the detection of patients with TB, but that the specificity of QuantiFERON is higher, especially in those individuals who have been vaccinated with BCG.
Let’s switch gears now and turn our focus to information that may be helpful to hospitals and clinical laboratories interested in using QuantiFERON In-Tube. There are multiple steps with this method that must be completed properly to ensure accurate performance. These steps include, collection of the blood specimen into the three tube collection set, storage of the specimen, incubation of the tube set, transport from the collection site to the testing laboratory, and finally, testing of the specimen. I want to stress that accurate results require accurate preanalytic processing. So let’s walk through the steps of this assay, and I’ll emphasize some key points for accurate performance along the way.
When discussing any topic related to tuberculosis, I think it’s always important to begin by underscoring the significant global impact of this disease. In 2007, the Centers for Disease Control and Prevention estimated that nearly 2 billion persons, or 1/3 of the world’s population, were infected with TB. Among those that are infected worldwide, approximately 9 million develop active TB disease and roughly 2 million die from TB each year. This disease is especially problematic in the HIV population, with recent data suggesting that 1/3 of HIV-infected patients are coinfected with TB, and that nearly 50% of deaths among HIV infected persons are attributed to TB. These figures are really quite staggering, and highlight why new and improved diagnostic methods are desperately needed to identify those with active and latent tuberculosis infection.
As I mentioned, the first step involves collection of whole blood into the specialized three tube set provided by the testing laboratory. It is extremely important to draw exactly 1-mL of blood into each of the 3 tubes. There’s a black mark on the side of each tube that indicates the 1-mL fill line. Although the valid range for this test is between 0.8 and 1.2 mL, I encourage laboratories to fill as closely to the fill-line as possible.
On the next slide, we see an example of a QuantiFERON tube set that has been correctly filled to the 1-mL fill line. However, the following images are examples of tubes that have either been under-filled, as seen with the tube on the left, or overfilled, as seen with the tube on the right. These tubes have been drawn incorrectly and this may lead to inaccurate results.
The second step in the process is mixing the blood collection set. Unlike most phlebotomy draws, the goal with the QuantiFERON In-Tube collection set is to mix the tubes by shaking vigorously for 5 seconds, or about 10 times. This allows the blood to resuspend the antigens that are dried on the side of the tubes.
After shaking, the entire inner surface of each tube should be coated with blood as you can see in the image on the right. In addition, proper shaking will lead to frothing of the blood, which is required for correct performance of the test.
In the next step, the tubes should be labeled with patient identifiers so that the colored QuantiFERON strip at the top of the tube, and the black fill line are not covered by the label.
The tubes should then be placed in a 37ºC incubator as soon as possible, and definitely within 16 hours of collection. However, I would encourage laboratories to place the tube set into the incubator as soon as they have been mixed and labeled. If for some reason you’re unable to incubate immediately, maintain the tubes at room temperature until they can be incubated. And if the tubes are not immediately placed at 37ºC, you should repeat shaking vigorously for 5 seconds immediately prior to incubating at 37ºC. One additional important note is that the tube set should not be refrigerated or frozen prior to incubation.
In the next step, incubate the tubes upright at 37ºC for 16 to 24 hours. Ensuring that the tubes are incubated at the correct temperature and for the correct amount of time is essential to the accurate performance of the test.
Following incubation, centrifuge the tubes using a swinging bucket for 15 minutes at 3000 RCF. This centrifugation step separates the plasma from the cell layer as you can see in the image on the right hand side of your screen.
In the final step, place all 3 tubes together in the QTB transport bag which will be supplied to you by Mayo Medical Laboratories. You’ll then ship the QTB kit at refrigerate temperature to Mayo, where the specimens will be tested. It’s important to note that the specimens should not been sent frozen, as this may negatively effect the results.
In conclusion, we’ve discussed how interferon gamma release assays, such as QuantiFERON, offer a number of advantages over tuberculin skin testing. These advantages include increased specificity, a one time blood draw with no return visit, and an objective result typically available within 24 hours.
Conventionally, the diagnosis of latent tuberculosis, or LTBI, has been accomplished through the use of the tuberculin skin test, which you may also hear referred to as the intradermal Mantoux test or the PPD. This test was originally derived from Koch’s tuberculin, and was first described by Koch in 1890. The tuberculin skin test has been in routine use since 1910, making it one of the oldest diagnostic methods still in use today.
The tuberculin skin test is useful for the identification of individuals who are asymptomatic but infected, but is also routinely used when examining patients with active TB.
In addition, QuantiFERON In-Tube allows for an extended transport time from the collection site to the testing laboratory.
I’d like to finish by once again emphasizing that accurate QuantiFERON results require accurate collection, storage, incubation, transport, and testing. Also, like any other serologic test, the results of QuantiFERON should not be considered diagnostic, but should be interpreted in the context of other clinical and laboratory findings.
Although the tuberculin skin test has been used routinely for nearly a century, there are significant limitations associated with this test that I’d like to review. First, the tuberculin skin test requires trained personnel to accurately administer the purified protein derivative, and placement errors can cause erroneous results.
In addition, the test is subject to reader variability, and this subjective interpretation can lead to varying and inaccurate findings. The tuberculin skin test is also subject to something called the “boost” response, where an initial placement of PPD can enhance or boost subsequent TST reactions.
One of the major limitations of the tuberculin skin test is its low specificity, especially in individuals that have received the BCG vaccine, or in those that are infected with certain nontuberculous mycobacteria. The test can also demonstrate low sensitivity, and this is a problem in immunocompromised hosts. The final limitation that I’d like to emphasize is the low compliance rate for patients returning to have their skin test read 48 to 72 hours following placement. This requirement for a second visit has ultimately led to many patients being “lost to follow-up” without their TB status being determined, and this increases the risk of patients with LTBI going unidentified.
Due to the limitations associated with the tuberculin skin test, there’s been a significant amount of work over the past decade put into the development of new assays for the diagnosis of active and latent TB.
Probably the most successful and well-studied approach have been the interferon gamma release assays. The general principle behind IGRAs is the measurement of in vitro levels of interferon gamma produced by sensitized T cells that have been stimulated by purified or synthesized TB antigens. In the first step, blood is collected and mixed with TB-specific antigens. The tubes are then incubated at 37°C for generally 16 to 24 hours. During this incubation period, antigen presenting cells process the purified TB antigens. Following processing, the antigen presenting cell then distributes the antigen on its surface for recognition by an antigen-specific T cell. If an individual has been exposed to TB in the past, the primed or sensitized T cell will then produce and release interferon gamma in response to binding to the antigen presenting cell. Following the incubation period, the tubes are centrifuged to separate the plasma and cell layers. And finally, the tubes are sent to a clinical laboratory where the levels of interferon gamma are measured by an ELISA-based assay.
Now that we have a general understanding for the principle behind this technology, I’d like to turn our focus to a particular interferon gamma release assay, called QuantiFERON TB Gold In-Tube, which we’ve recently implemented into our laboratory at Mayo Clinic. QuantiFERON is an in vitro whole blood test that is designed for use as an aid in diagnosing both latent and active TB disease.
There have been several versions of QuantiFERON since its initial release in 2001, but the latest version, known as QuantiFERON In-Tube, received FDA clearance in October of 2007. This 3rd generation assay offers several advantages over its predecessors, including an extended transport time from the collection site to the testing laboratory. QuantiFERON In-tube uses three peptide simulating antigens, including ESAT-6, CFP-10, and TB7.7, for the stimulation of T cells and subsequent secretion of interferon gamma.
Unlike the first two generations of QuantiFERON, In-tube utilizes a specialized, 3 tube collection set. The first tube has a gray top, and is called the Nil tube. The Nil tube serves as the negative control, and no interferon gamma production, or very limited interferon gamma production, should be observed with the Nil tube. The second tube has a red top and is the TB-antigen tube. The antigen tube is coated with the lyophilized TB antigens ESAT-6, CFP-10 and TB 7.7. These antigens, when mixed with blood containing sensitized T cells, will lead to the production and secretion of interferon gamma. And finally, the third tube is called the Mitogen tube, and it has a purple top. The Mitogen tube contains phytohemagglutinin, which acts as a positive control for the production of interferon gamma from T cells.
Following a successful run, the results of QuantiFERON In-Tube are reported out as positive, negative, or indeterminate. An indeterminate result implies that the level of interferon gamma detected by the ELISA was between the cutoff for positive and negative. An indeterminate result may be due to several factors. First, it may be due to a low mitogen (or positive control) response. This can occur in immunocompromised patients, or from improper mixing of the collection tubes. The second scenario which may result in an indeterminate is a high nil (or negative control) value. This may result from improper collection, incubation, transport or processing of the collection tubes.
Now that we’ve discussed some general background points to QuantiFERON, you may be asking
“What are the advantages of this method over the tuberculin skin test?”
First, QuantiFERON In-Tube requires basic phlebotomy, whereas the TST is subject to placement error.
Second, the results of QuantiFERON are typically available within 24 hours, while the results of TST require 48 to 72 hours.
Third, a single visit is required for QuantiFERON. In contrast, a return visit is required for the TST, and as I mentioned earlier in the presentation, this may lead to a substantial number of patients who fail to have their TST interpreted and their TB status may remain unknown.
A fourth advantage of QuantiFERON is that the results are objective, while the TST requires a subjective interpretation that may vary between readers.
Other advantages of QuantiFERON are that this is an in vitro test, so no boost response is observed. In contrast, the TST is an in vivo approach, and an initial placement of PPD may boost or enhance the results of subsequent skin tests.
A final advantage that I’d like to discuss relates to the specificity of QuantiFERON. This assay does not seem to be affected by prior vaccination with BCG or infection with most nontuberculous mycobacteria. In contrast, false positives skin tests may be observed in patients vaccinated with BCG or infected with other mycobacteria.