By Renee DiIulio

 By 2005, the World Health Organization (WHO) in Geneva, Switzerland aims to detect 70% of new infectious tuberculosis (TB) cases and to cure 85% of those detected. In 2002, 18 countries had achieved these goals, but globally, only 37% of the estimated number of patients had received treatment. Left untreated, each person with active TB will infect an average of between 10 and 15 people every year.

For roughly a century, the preferred method of screening for TB has been the Mantoux tuberculin skin test (TST), but its subjectivity and cross-reactive features can bring controversy to its results. The WHO guidelines recommend diagnosis with a microbiological examination, which detects the presence of Mycobacterium tuberculosis on a sample smear, most often created with sputum. Both tests are inconvenient for patients and can require confirmation with a culture. The Mantoux testing procedure requires patients to return between 48 and 72 hours after the initial injection to have results read. Moreover, the sputum specimens needed for the microbiological exam can be difficult to produce. The tests also have issues with false readings.

For these reasons, Cellestis International of Carnegie, Australia believes its QuantiFERON-TB Gold (QFT Gold), a newer version of QuantiFERON TB (QFT) that is currently undergoing review at the US Food and Drug Administration (FDA) can play a role in helping the WHO achieve its goals. Though it does not deliver results at the point of care, the test offers more accuracy with greater speed and fewer costs than the skin test. It produces results in less than half the time and matches some of the aims of many POC products. The WHO has also considered the test a possible replacement for the microbiology procedure.

Two TB-Specific Proteins Eliminate Cross-Reaction
In the guidelines published on its Web site, the Centers for Disease Control and Prevention (CDC) in Atlanta compares the first version of the QFT test to the skin test. The guidelines state: “As a diagnostic test, QFT 1) requires phlebotomy; 2) can be accomplished after a single patient visit; 3) assesses responses to multiple antigens simultaneously; and 4) does not boost anamnestic immune responses. Compared with TST, QFT results are less subject to reader bias and error.”

 The Mantoux intradermal tuberculin skin test for tuberculosis is read positive when the tuberculin reaction exceeds a certain diameter determined by patient-specific risk factors. (Source: CDC)

A skin-test reading is positive when the tuberculin reaction exceeds a certain diameter determined by patient-specific risk factors. In addition to the subjectivity of the reader, the test may produce false-negatives if the patient has been screened too soon after being exposed; it may also register false-positives if the patient has been immunized with bacille Calmette-Guérin (BCG) or exposed to environmental mycobacterium.

The microbiological test for TB permits only the presumptive diagnosis of TB because the acid fast bacilli (AFB) in stained smears may similarly be mycobacteria other than M. tuberculosis. In addition, false-negatives are also a risk; an infected smear may not include enough bacteria.

QFT Gold eliminates many of these cross-reactive effects by identifying the presence of T-cells specific for TB infection. According to Tony Radford, PhD, managing director and CEO of Cellestis, improvements to the QFT TB product were made through the use of the TB-specific antigens ESAT-6 and CFP-10, which are made only by M. tuberculosis. The specificity helps to eliminate cross-reactions with organisms that may cause false-positives in the skin test, such as the tuberculosis vaccine organism BCG and most other environmental mycobacteria.

Studies have found QFT Gold exhibits specificity greater than 98% and sensitivity at about 90%. The test produces results within 24 hours (181/2 to be exact) and is performed on whole blood.

Laboratories performing the test incubate small amounts of the undiluted whole blood with the TB-specific proteins overnight and determine the Interferon-gamma (IFN-gamma) levels in the plasma the next day. A person is considered positive for M. tuberculosis infection if he registers an IFN-y response to either ESAT-6 or CFP-10 above the test cutoff. No risk stratification or patient history needs to be applied to the interpretation of the result, unlike the skin test.

“The current skin test has varying catalyst points, and everyone does it differently. The QFT Gold test provides a yes or no answer, no subjectivity involved,” says Radford.

The QFT Gold test has a CE marking for use in Europe and approval in Australia. Studies are under way in Holland, the United Kingdom, Italy, and Spain. Approvals are being awaited in the US and Japan. An answer from the FDA is expected fairly soon, says Radford. The request for approval was submitted in December and was not expected to extend beyond 180 days.

Test Crosses Animal/Human Barrier
The older version of the test sailed through all approvals. Its basic technology was originally developed for TB testing of cattle in Australia. “The skin test used on cattle then was proven inadequate, and the TB control campaign needed a better test. The Cellestis technology is derived from what was determined to be the best platform,” recounts Radford.

The transition from cattle to humans was made with a switch in proteins, to those pulled from the human tuberculin rather than the bovine version. The underlying technology is the same. The company was founded in 2000 to develop and market QuantiFERON products.

The procedure is a patented whole blood method, which detects cell-mediated immunity (CMI) responses. Details, available on the company Web site, state “the QuantiFERON test takes advantage of the fact that individuals primed in vivo with exogenous or endogenous antigen have lymphocytes in their blood that maintain an immunological memory for the priming antigen. Addition of antigen in vitro to blood taken from primed individuals results in rapid restimulation of antigen-specific effector/memory T cells and the release of the cytokine IFN-gamma, which is used as a specific marker for a cellular immune response mounted to the antigen (recall response). Stimulation of effector/memory T cells in whole blood with specific antigen(s) or mitogen and subsequent quantification of IFN-gamma by a rapid, single-step ELISA forms the basis of the QuantiFERON test.”

Improvements Save Time and Money
The QuantiFERON process eliminates the booster effects possible with the skin test and creates a highly reproducible in vitro procedure. The use of ESAT-6 and CFP-10 make the QFT Gold test highly specific for low-risk populations and more sensitive in determining active TB cases, according to Radford.

The test is unaffected by “nearly all tuberculous mycobacteria” and therefore is more accurate in populations that have been vaccinated with BCG or impacted by environmental factors.

The CDC reports that in the US, foreign-born persons accounted for 41.7% of the TB cases in 2001, a rate 8.7 times greater than the national figure. “The predominant test groups in Canada and the US are immigrants, at about 54%. These are the same people who have most often been immunized with BCG,” says Radford. In addition, he cites studies that suggest roughly half of the responses to the TB skin test in the US may be due to environmental bacteria, potentially creating a 50% false-positive rate. To eliminate the cross-reactions from either source creates economies in both time and expense for the patient and physician as additional unnecessary testing and treatment may be avoided.

In addition, the test procedure creates savings as well. Because the patient need not return for a second visit, less office time is required from the physician. The patient, too, saves time and money, and the population at large benefits because many patients will not show up the second time, particularly if they do not believe they are ill.

TB bacteria can lie dormant within a person for years, manifesting when triggered or when the host’s immune response has weakened. The WHO estimates that 5%–10% of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life. Patients with compromised immune systems, such as those with HIV or organ transplants, are at an increased risk of contracting the disease. Once the illness has been activated, the person becomes contagious, spreading the TB bacteria via the air. A person need only inhale a small amount of bacteria to become infected.

The WHO estimates that someone in the world is newly infected with TB every second, and that one third of the world’s population is currently infected. The organization approximates that in 2002, 2 million deaths could be attributed to TB. As more of these cases feature drug-resistant versions of the disease, the greater a threat it presents to all nations and the more urgent the desire to eradicate it. The QuantiFERON-TB Gold test aims to become a tool in the arsenal of the medical community that will help it win this battle.

For additional information, contact Tony Radford, managing director and CEO, Cellestis International, Carnegie, Australia; phone: 61 41953 0785; email: [email protected];  or Jill Hoffman, senior account executive, FischerHealth; (310) 577-7870 ext. 113; email: [email protected].

Renee DiIulio is a contributing writer for Clinical Lab Products.