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The diagnostic of tuberculosis

Poster-Tuberculosis-ENWhat is tuberculosis?

Worldwide, tuberculosis is one of the 10 leading causes of death from an infectious agent, ahead of HIV. In 2019, it was estimated that 1.4 million people died from tuberculosis and 10 million people contracted the disease. For patient with HIV, tuberculosis is the leading cause of their death.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis, which mostly infects the lungs. It is transmitted through the air, by the projection of bacteria when sneezing or sputtering while talking. Symptoms of tuberculosis are fever, fatigue, a fatty cough, and night sweats. This is a curable disease by the combination of 3 antibiotics during 6 months.

There are several stages of tuberculosis: we can distinguish the latent tuberculosis, which is not contagious, from the active tuberculosis, which is contagious. The latent tuberculosis is, in 85% to 95% of the cases, controlled by the body. The remaining 5% to 15% of the cases correspond to the active tuberculosis or the “tuberculosis disease” responsible for clinical and / or radiological symptoms.

The means of tuberculosis diagnosis

Several types of diagnosis are used to guide and confirm the disease. First, the clinical diagnosis passes through the detection of symptoms such as cough accompanied by sputums, fever, a deterioration of the general condition as well as a weight loss and night sweats. The patient’s history should also be considered, especially if the patient travelled in a country at risk.

X-rays of the lungs may also show abnormal opacities suggestive of the disease.

The immunological tests used today represent diagnostic aid to detect carriers of latent tuberculosis. This can evolve into an active form of tuberculosis.

The diagnosis allowing to confirm the disease, to identify the mycobacterium and its possible resistance to antibiotics for a treatment administration is the bacteriological diagnosis. This includes several analyzes: microscopy, PCR (Polymerase Chain Reaction), culture, antibiograms…

 

Means-diagnostics

 

Review of the main bacteriological methods to diagnose tuberculosis

Depending on the sample as well as epidemiological, technical, and economic factors, the tests performed will not be the same. Therefore, each country follows its own recommendations. Despite the number of diagnosis methods which are steadily increasing, the reference method for the diagnosis of tuberculosis remains the culture of biological samples.

The first-line method to see tuberculosis is the microscopy. For many decades, direct microscopic examination preceded by staining biological samples has made it possible to observe  the presence of acid-fast bacilli (AFB). It is a simple, fast and inexpensive test providing information on the AFB concentration of the patient. The reference stain is the Zielh-Neelsen (SN) staining, showing pink AFP on a blue background (cells). Auramine fluorescent staining is also widely used in laboratories. It allows the observation of AFB possible as they appear fluorescent green on a red background (cells) with more sensitivity than the ZN staining. However, this staining requires specific equipment: a mercury lamp microscope or a LED microscope. The sensitivity of this method varies according to the type of sample: 65% for pulmonary samples and 10% to 20% for extra-pulmonary samples. 

Colorations-tuberculosis-en

 

Culture is the most sensitive and specific test for mycobacteria. Despite the slow growth of the mycobacteria tuberculosis complex, the diagnosis of tuberculosis is based on their culture. Sample culture is generally preceded by a digestion and decontamination of pulmonary samples such as sputum. The combination of solid and liquid cultures is the most efficient method with a very low detection threshold: 10 to 10² bacilli / mL of sample. On solid media it will take 3 to 6 weeks to detect an AFB, depending on the mycobacterial concentration. For liquid media, the positivity period is generally between 10 and 14 days.

The antigenic method by immunochromatography consists in looking for a protein specific to the mycobacteria of the tuberculosis complex, the MPT64 protein. This test is done following a positive culture on a solid or liquid medium and highlights the tuberculosis complex in 15 minutes.

The identification of mycobacteria can also be done by mass spectroscopy. This technique is carried out from cultures and allows to identify both the mycobacteria of the tuberculosis complex as well as most atypical mycobacteria.

PCR methods allow rapid and very sensitive results. These methods can highlight the presence of mycobacteria of the tuberculosis complex, but also a resistance to anti-tuberculosis drugs.

Among these PCR methods, the GeneXpert® technique is widely used. This is a reliable and fast method to detect mycobacteria of the tuberculosis complex, but also a potential resistance to rifampicin. Rifampicin is a major antibiotic for the tuberculosis treatment. This PCR provides a diagnosis within 2 hours.

 

Conclusion

The sensitivities and specificities of the different methods depend on the origin of the biological sample (intra or extra-pulmonary) as well as the mycobacterial concentration. Each technique thus has its advantages and limitations.

The advent of PCR techniques represent an important advance in the diagnosis of tuberculosis. However, these techniques are expensive and require staff training. PCR tests are particularly recommended as a first-line intention in areas where tuberculosis is endemic. Despite the sensitivity of this technique, depending on the case, it still requires additional tests. When the PCR result is negative, it does not rule out tuberculosis. In areas not endemic to tuberculosis, the microscopy test is therefore recommended as a first-line intention associated or not with PCR. Culture then remains the essential confirmation step for tuberculosis diagnosis.

In this field, RAL Diagnostics has developed effective staining products that simplify the work of technicians with the TB Prep, Fluo-RAL and Cold ZN kits, thus Stainer Fluo-RAL, Cold ZN and Fluo-RAL Methylene Blue kits.

Download the poster on diagnostic methods for tuberculosis: Poster-Tuberculosis-EN

It is also available in the Resources area.

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Bibliographical references:

Société Française de Microbiologie, 2015, Référentiel en microbiologie médicale, 5ème édition 2015

L. Slim-Saidi, E. Mehiri-Zeghal, A. Ghariani, F. Tritar, 2015, Nouvelles méthodes de diagnostic de la tuberculose

C. Guillet-Caruba, V. Martinez, F. Doucet-Populaire, 2014, Les nouveaux outils de diagnostic microbiologie de la tuberculose maladie