Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice
in the Department of Laboratory Medicine and Pathology at Mayo Clinic.
Did you know that according to the World Health Organization, tuberculosis is one of the top
10 causes of death worldwide, and that about 25% of the world’s population is infected with
latent TB?
Well, due to the significant prevalence of this disease, it’s essential that we have
good laboratory tools to identify patients who are infected, so that we can treat the
illness and help reduce the spread of TB.
In this month’s Hot Topic, my colleague, Dr. Elli Theel, will discuss a new interferon-gamma
release assay that can help identify patients that are infected with TB.
I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing
Mayo Clinic the opportunity to partner in your patients’ healthcare.
Thank you for the introduction and also to the viewers for joining us today.
Before I begin, just a note that I do not have any relevant financial disclosures to
share.
As you view the following presentation, consider the following important points regarding testing.
How is this testing going to be used in your practice?
When should the tests be used, and how will results impact patient management?
And also, specifically for the new QuantiFERON-TB Gold Plus assay that I will be discussing,
consider when and how to plan for transitioning to this new interferon-gamma release assay
at your institution, keeping in mind that the current QuantiFERON-TB Gold In-Tube assay
will be discontinued by the manufacturer in June of 2018.
So, today I will be talking about the new FDA-cleared QuantiFERON-TB Gold Plus assay
from Qiagen, which, similar to the current QuantiFERON-TB Gold In-Tube assay, is intended
to be used for the indirect detection of infection with Mycobacterium tuberculosis.
First, however, I wanted to quickly review the evolution of laboratory diagnostics for
tuberculosis (TB), which, similar to the growth of the organism itself, has been fairly slow
until recently.
Briefly, following identification of Mycobacterium tuberculosis or Koch’s bacillus in 1884,
the tuberculin skin test was developed in the early 1900s, which involves intradermal
injection of a purified protein derivative from an attenuated Mycobacterium tuberculosis
strain, followed by assessment for the presence or absence of a delayed-type hypersensitivity
reaction at the injection site 48 to 72 hours later.
Almost 90 years after that in the late 1990s and early 2000s, we were still primarily relying
on the skin test for detection of latent tuberculosis and on direct smears and culture for detection
of active disease.
In the last 10 to 15 years or so, diagnostic testing for TB sped up a bit, with the development
of specific probes for direct testing of colonies, and we now have direct sequencing and PCR
assays available, which can be done directly off of patient samples.
And while these methods are great for diagnosis of active tuberculosis, they are not useful
for detection of latent infection.
In an effort to improve in this particular area, interferon-gamma release assays (or
“IGRAs”) were developed and released in the early 2000s.
Until recently, there were just two IGRAs with FDA-clearance in the United States, including
the T.SPOT-TB assay from Oxford Immunotec and the QuantiFERON-TB Gold In-Tube ELISA
manufactured by Qiagen.
In June of 2017, Qiagen released the newest version of the QuantiFERON-TB assay, referred
to as the QuantiFERON-TB Gold Plus, or QFT-Plus assay.
Regardless of the manufacturer, IGRAs are essentially indirect markers for exposure
to Mycobacterium tuberculosis and are based on the concept that individuals who are infected
with TB have primed memory T cells, which, upon re-exposure to specific TB antigens,
will be activated and stimulated to release IntraFERON-gamma.
This IntraFERON-gamma is then detected by a standard ELISA in the case of the QuantiFERON
assays or by an enzyme-linked immunospot (ELISPOT) method in the T.SPOT assay.
For the purposes of today’s presentation, I will just focus on the new QuantiFERON-Plus
assay, which was developed with the goal to increase the sensitivity of this assay for
detection of both active disease and latent infection.
To do this, the manufacturer included TB peptides to stimulate both CD4- and CD8-positive T
cells in the QuantiFERON-Plus assay, and this differs from the current QFT-Gold assay, which
contains TB peptides able to stimulate CD4 T cells only.
So, why are both of these cell types important?
Well, CD4 T cells have always been known to be essential for TB control.
Upon stimulation, they secrete cytokines to regulate and assist the immune response, primarily
through secretion of IntraFERON-gamma to activate macrophages.
More recently, CD8 natural killer T cells have also been identified as critical for
control of TB.
They, likewise, secrete IntraFERON-gamma, but they have also been shown to directly
kill TB-infected cells, and they can lyse intracellular TB bacilli as well.
What is the difference between the QFT-Gold and the new QFT-Plus assays?
Well, first, while the QFT-Gold assay has 3 tubes, the QFT-Plus assay has 4.
The mitogen and nil tubes are identical between the two tests, and, as a reminder, the mitogen
tube contains phytohemaglutanin, which serves as a positive control for T-cell activity,
while the nil tube essentially measures the level of background or circulating IntraFERON-gamma
in the patient.
As you can see, however, a notable difference between the two versions is that the QFT-Plus
has two TB antigen tubes, labeled TB 1 and TB 2, versus the single TB antigen tube in
the QFT-Gold assay.
The composition of the TB antigen tubes is notably different between the two versions.
First, while the QFT-Gold assay has peptides from the Mycobacterium tuberculosis ESAT-6,
CFP-10 and TB7.7 proteins, the TB7.7 antigen has been removed entirely from the QFT-Plus
assay.
The three TB antigens in the QFT-Gold assay were designed as long peptides able to be
recognized by MHC class II molecules for presentation to CD4-positive T cells, and aside from removal
of the TB7.7 antigen, the TB Antigen 1 tube in the QFT-Plus assay has the same long peptides
for stimulation of CD4-positive T cells.
The TB Antigen 2 tube, however, has both long and short peptides for ESAT-6 and CFP-10 for
presentation on both MHC class II and class I molecules for stimulation of CD4- and CD8-positive
T cells, respectively.
Another key difference between the two QuantiFERON versions is that for the QFT-Plus test, whole
blood can be collected directly into the four separate tubes, as is the current protocol
for the QFT-Gold assay, or into a single lithium heparin-tube, which can then be aliquoted
into the four separate tubes in the laboratory.
Importantly, Mayo Medical Laboratories will only accept processed QFT-Plus tubes for testing,
as is currently the process for the QFT-Gold assay.
Lithium-heparin tubes submitted from MML clients will not be accepted due to strict incubation
time and temperature requirements by the manufacturer, which may not be adhered to during the shipping
and transport process.
For more information on specimen collection, handling, and processing of lithium-heparin
tubes for the QFT-Plus assay, please refer to the manufacturer instructions or contact
Mayo Medical Laboratories for more information.
Following collection of patient whole blood, processing of the QFT-Plus tubes is identical
to processing of the QFT-Gold tubes.
As a quick review, anywhere from 0.8 to 1.2 ml of blood should be collected or aliquoted
into each of the four separate QFT-Plus tubes.
The tubes are then mixed by firmly inverting 10 times and incubated at 37 degrees for 16
to 24 hours.
And it is during this time that the IntraFERON-gamma is being produced, first with the antigen-presenting
cells taking up and processing the antigens and then presenting them to the antigen-specific
T cells, which, if present, will release IntraFERON-gamma.
Following the incubation, the tubes are spun to collect the plasma, which can be stored
for 28 days at 2 to 8 degrees Celsius prior to testing.
And at Mayo Medical Laboratories, the QuantiFERON and IntraFERON-gamma ELISAs are performed
on the Dynex Agility instruments.
When it comes to result calculation, qualitative cutoff values, and result interpretation,
the QFT-Plus is identical to the QFT-Gold assay, with the key exception that if either
one or both of the QFT-Plus TB antigen tubes are equal to or greater than 0.35 IU/mL and
are at least 25% of the nil tube value, the patient is considered positive.
In summary, the new QFT-Plus assay for indirect detection of TB received FDA clearance in
July of 2017 and is intended to improve upon the sensitivity over the current QFT-Gold
version of the assay.
The QFT-Plus differs from the QFT-Gold in that it will stimulate both CD4- and CD8-positive
T cells, and current literature suggests non-inferior performance of the QFT-Plus assay compared
to the QFT-Gold version, though additional studies in immunosuppressed patients in others
are needed and ongoing.
Importantly, the current QFT-Gold assay will be discontinued by the manufacturer in July
of 2018, and therefore, Mayo Medical Laboratories has begun the process to convert testing to
the QFT-Plus version of the assay, and it will begin performing this test on February
13 of 2018.
Following this date, the QuantiFERON-Gold test will no longer be orderable.
In an effort to make this conversion as seamless as possible for our patients and MML clients,
QFT-Plus tubes will be available for order on January 13, 2018.
Clients are encouraged to begin decreasing their QFT-Gold tube supply and begin ordering
the QFT-Plus tubes around this time, in addition to begin considering whether whole blood will
be collected into a single lithium-heparin tube or whether patient samples will continue
to be collected directly into the QFT-Plus tubes.
Again, as a reminder, MML will not accept lithium-heparin tubes or unprocessed QFT-Plus
tubes.
For any questions and assistance during the conversion process, clients are encouraged
to contact Mayo Medical Laboratories.
Thank you for your attention.