Download Mycobacterium Tuberculosis Bacilli (MTB) Rapid and specific testing

Mycobacterium Tuberculosis Bacilli (MTB) Rapid and specific testing kit to diagnose and distinguish
latent and active stages.
Full Description:
Investment in the tuberculosis diagnostics pipeline should remain a major priority for funders and
researchers. Mycobacterium Tuberculosis Bacilli (MTB) kills 1.5 million people every year, and is the
major killer of people living with HIV because of their weakened immune system. The TB disease is
developed in a two stage process. Stage 1, known as latent TB, a person is first infected with TB bacteria.
This phase can last for a very long time up to many years without feeling or looking sick. Stage 2, known
as active TB or TB disease, the bacteria have reproduced sufficiently to usually cause the person to have
become sick and seek treatment. In order to attain a diagnosis of active TB it must be confirmed that
there is definite evidence of TB bacteria in the body. Tuberculosis (TB) germs (bacteria) are spread from
person to person through the air. When someone with active, infectious TB disease of the lungs or
airways breathes out (or coughs, sneezes, sings, plays a wind instrument or sometimes even just talks),
tiny droplets containing TB germs are released into the air. These droplets can stay in the air for hours.
Thus, some of the TB diagnostic tests look directly for TB bacteria whereas others, such as the chest Xray, look for the effect of the bacteria on the lungs, a place where the bacteria usually reside. Improving
TB tests by providing an early and effective diagnosis of TB to ensure more lives are saved is a priority
for the medical community. Rapid, accurate, less costly tests that are easy to administer, using blood as
the clinical sample are needed. New detection platforms such as testing for multiple markers in blood,
improve sensitivity and specificity because it is possible to differentiate latent infection and active
disease.
There are several issues with the diagnosis of TB using the present diagnostic tests. There are different
tests for different types and phases of TB, so multiple tests may be needed for an accurate diagnosis. A
positive TB skin test or TB blood test only tells that a person has been infected with TB bacteria. It does
not tell whether the person has latent TB infection (LTBI) or has progressed to TB disease. Other tests,
such as a chest xray and a sample of sputum, are needed to see whether the person has TB disease.
There is chance for false negatives due to the bacteria not multiplying yet and with many tests there are
also false positives if someone has taken the vaccine or been exposed but not contracted TB. A negative
result for either test means that it is likely that the person tested does not have a TB infection. However,
it does not entirely rule out tuberculosis. It may mean that the person's immune system has not
responded to the antigen in the test or that it is too early to detect exposure. It takes about 6 weeks
after infection before a person demonstrates a positive reaction to TB screening tests. These tests are
time consuming, extensive and may not diagnose accurately. Thus, Testing TB can be expensive,
ineffective and time consuming.
Due to the time and expense of TB tests, individuals are only tested if they have certain risk factors such
as: have had frequent close contact with someone who has active TB disease, have lived in a country
where many people have TB, work or live in a nursing home, clinic, hospital, prison, or homeless shelter,
or have HIV infection or your immune system is not very strong, health care workers and other
professions that are brought close to populations that may be prone to TB, those who have been close
to people diagnosed with TB, those who are exhibiting the signs and symptoms of active TB, those who
use illegal drugs, when an individual has lived for an extended time in a country where TB is more
common, or yearly for healthcare workers at high risk. TB screening tests are also used sometimes as
part of a routine examination prior to starting school or a new job. Since mothers can pass TB to their
unborn children, pregnant women are sometimes screened. Children less than 5 years old should have
the TB skin test instead of the TB blood test as it is not known what side effects there are for this
population.
The Tests
The two most popular first choices for testing TB is a Tuberculin skin test (TST) or an interferon gamma
release assay (IGRA). Secondary tests are most often performed as well to determine what stage the
infection is in. Most people with a positive TB blood test have latent TB infection. A chest x-ray and
other tests are done to see if it is a latent TB infection or active TB disease. Both phases of TB can be
treated with medicine. However, without treatment, latent TB infection can become active TB disease.
The BCG vaccine Bacille Calmette-Guérin (TB vaccine given to children) may help protect young children
from getting very sick with TB but this protection goes away as they get older and they still can get
latent TB infection and active TB disease. These individuals must be given the TB blood test because it
will be “positive” only if you have TB germs whereas the skin test will always be positive regardless of
condition.
The type of test may depend on the type of TB suspected:
latent TB
TB Skin Test
Several tests can be used to confirm a diagnosis for latent TB, if you have no symptoms but have been in
close contact with someone infected or in a high risk category. The Tuberculin skin test, Mantoux test,
involves the injection of a small amount of purified protein derivative (PPD) solution under the first layer
of skin of the forearm and an evaluation of the injection site conducted by a health practitioner at 48
and/or 72 hours to see if a local skin reaction has occurred.
Problem: Once positive (due to exposure or vaccine), a TST reaction will usually remain positive and the
skin reaction to subsequent tuberculin skin tests may become increasingly severe. You may also need a
chest x-ray to confirm diagnosis. If you have had the BCG vaccination, you may have a mild skin
reaction to the Mantoux test, and be misdiagnosed. The problem with using it in countries with high
rates of TB infection is that the majority of people may have latent TB. The TB skin test result depends
on the size of the raised hard area or swelling, and the larger the size of the affected area the greater
the likelihood that the person has been infected with TB bacteria at some time in the past. But
interpreting the TB skin test result, that is whether it is a positive result, may also involve considering
the lifestyle factors of the person being tested for TB.2 The TB skin test also cannot tell if the person has
latent TB or active TB disease.
IGRA Test
The IGRA test measures the release of a substance called gamma interferon by white blood cells in a
sample of blood when the cells are exposed to specific TB Antigens. A positive Mantoux test negates an
IGRA test to confirm.
Problem: The test requires viable white blood cells, so the IGRA blood sample must be received and
tested by a laboratory within a designated window of time or the test is no good. If the BCG vaccine was
given then the test will be a false positive. Occasionally, a person infected with or exposed to
other Mycobacterium species, for example Mycobacterium kansasii, will give a false-positive TST or
IGRA result for TB. Positive results must be followed up by other tests such as chest X-rays to look for
signs of active TB disease. If active TB disease is suspected, Patients can have different antibody
responses suggesting that they have active TB even when they do not. Antibodies may also develop
against other organisms which again could wrongly indicate they have active TB. In addition, different
organisms share the same antigens, making tests results unreliable. These factors can result in TB
disease not being identified or wrongly diagnosed.
BCG Vaccine
BCG is not used as a vaccine in the United States, but it is often routinely administered in countries with
a higher incidence of TB and it is used in the U.S. as a treatment for some cancers. However, in countries
where its given to children, the resistence goes away when they are older and they may contract TB.
Also, the skin test may test positive when older even if they don’t have TB but the blood test will not
unless they actually have TB.
Tests for Active TB
Pulmonary TB
A diagnosis of pulmonary TB can be difficult and several tests are usually needed. This includes a chest xray to look for changes in the appearance of your lungs that are suggestive of TB. Samples of phlegm will
also often be taken and checked for the presence of TB bacteria.
Problems: This is expensive and time consuming and requires technical expertise.
Extrapulmonary TB
Several tests can be used to confirm a diagnosis. Including: a computerized scan (CT), magnetic
resonance imaging scan (MRI), ultrasound scan, urine and blood tests, a biopsy, or a lumbar puncture.
The lumbar puncture takes cerebrospinal fluid (CSF) to look for TB infection in the central nervous
system.
Problem: This testing is expensive and time consuming and requires specialized equipment and
technical expertise.
X-ray and CT scans
Acute pulmonary TB can be easily seen on an X-ray. CT shows more detail.
Problem: the picture it presents is not specific and a normal chest X-ray cannot exclude extra pulmonary
TB. Also, in countries where resources are more limited, there is often a lack of X-ray facilities
Sputum
Smear microscopy of sputum is often the first TB test to be used in countries with a high rate of TB
infection. Sputum is a thick fluid that is produced in the lungs and the airways leading to the lungs, and a
sample of sputum is usually collected by the person coughing. A very thin layer of the sample is placed
on a glass slide, and this is called a smear. A series of special stains are then applied to the sample, and
the stained slide is examined under a microscope for signs of the TB bacteria. Sputum smear microscopy
is inexpensive and simple, and people can be trained to do it relatively quickly and easily. In addition the
results are available within hours. The sensitivity though is only about 50-60%.
Problems: In countries with a high prevalence of both pulmonary TB and HIV infection, the detection
rate can be even lower, as many people with HIV and TB co-infection have very low levels of TB
bacteria in their sputum, and are therefore recorded as sputum negative.
Fluorescent microscopy
The use of fluorescent microscopy is a way of making sputum TB tests more accurate. With a fluorescent
microscope the smear is illuminated with a quartz halogen or high pressure mercury vapour lamp,
allowing a much larger area of the smear to be seen and resulting in more rapid examination of the
specimen.
Problems: One disadvantage is that a mercury vapour lamp is expensive and lasts a very short time.
Such lamps also take a while to warm up, they burn significant amounts of electricity, and electricity
supply problems can significantly shorten their life span.
Using culture to test for TB
Culturing is a method of studying bacteria by growing them on media containing nutrients. Culturing and
identification of M. tuberculosis provides a definitive diagnosis of TB and can significantly increase the
number of cases found. Culture can also provide drug susceptibility testing, showing which TB drugs a
person’s bacteria is resistant to.
Problems: Culture is much more complex and expensive than microscopy to perform as it requires
specific equipment and enhanced laboratory facilities. Diagnosing TB using culture can also take weeks
because of the slow growth of TB bacilli. It averages 4 weeks to get a conclusive test result using the
most common methods of solid media, with another 4-6 weeks to produce drug susceptibility results.
Serological or blood tests for TB
Serological tests for TB are tests carried out on samples of blood, and they claim to be able to diagnose
TB by detecting antibodies in the blood. However, testing for TB by looking for antibodies in the blood is
very difficult. TB Blood tests approved by FDA and available in the US include: QuantiFERON-TB Gold inTube test and T-Spot. They are good to give if you had a negative skin test or the BCG vaccine and are
suspected of having TB. They do not help differentiate latent tuberculosis infection (LTBI) from
tuberculosis disease.
Problem: As a result serological tests, sometimes called serodiagnostic tests, for TB are inaccurate and
unreliable, Patients can have different antibody responses suggesting that they have active TB even
when they don’t. Antibodies may also develop against other organisms which again could wrongly
indicate they different organisms share the same antigens, making tests results unreliable and providing
a wrong diagnoses.
Molecular test
The Xpert MTB/RIF assay is a novel molecular, easy-to-use assay, which can lead to tuberculosis
identification in less than 2 h using several types of fluids, including blood. A recent study concluded that
Xpert MTB/RIF assay has high sensitivity and specificity for both pulmonary and extrapulmonary
specimens. This test is still in development.
The Challenge
The seeker specifically wishes to identify a robust method that:
1.
2.
3.
4.
5.
6.
Uses blood samples preferably, but other fluids will be considered if it is minimally invasive.
Minimizes the time for diagnosis to hours.
Easy to use and cost effective and can be done at point of care or reference lab.
Freedom to practice: no IP, however licensing opportunities will be considered.
Can detect and distinguish both latent and active TB.
Can detect TB in individuals who have had BCG test without false positives.
In Scope
1. Protocols that describe assays that include procedural steps with reagents and instruments.
2. Scientific explanations and reasons for the recommended techniques.
3. Reference to papers or clinical trials supporting the techniques.
Out of Scope
The Seeker is not interested in a solution that:
• Consists of a literature survey
• Requires excessively expensive reagents or supplies
• Has many or time-consuming steps
• Is primarily theoretical and would be difficult to assess efficacy in a practical manner
• Has negative health and safety issues or environmental impact
Criteria that must be met:
1. Level of detail provided for the proposed protocol such as reagents, solution formulations,
consumables, equipment
2. Applicability to high throughput, automated processing of up to a million embryos per month
3. Accessibility and cost of the technological solution, i.e., does not require development of an entirely
new technology.
4. Ease of method for use at the point of care
Project Deliverables
Challenge solutions should provide:
1. A detailed description and explanation of the proposed solution or solutions;
2. Persuasive scientific rationale and, ideally, experimental data, to justify the solution; and,
3. Full disclosure of any third party intellectual property used in the solution.
References:
CDC _ TB _ Fact Sheets _ Interferon-Gamma Release Assays (IGRAs)
CDC _ TB _ Testing & Diagnosis _ General Information about TB Blood Tests
CDC _ TB _ Testing & Diagnosis
Advances in tuberculosis diagnostics: the Xpert MTB/RIF
assay and future prospects for a point-of-care test
Evaluation of Xpert MTB/RIF assay for rapid molecular
diagnosis of tuberculosis in a two-year period in Croatia