Diagonostics

Latent TB infection Diagonostics

Tuberculin Skin Test (TST)

TST is an in vivo test whose aim is diagnosing latent tuberculosis infection (LTBI). It has been used for a hundred years in clinical practice and until few years ago it was the only available tool for LTBI diagnosis. It is commonly used as a complementary test for diagnosis of active TB.

It consists of a mixture of antigens obtained from a M. tuberculosis culture, called PPD (purified protein derivative), which is inoculated in the forearm by the Mantoux method. If the individual was previously exposed to M. tuberculosis, a cell-mediated immunity in the form of delayed-type hypersensitivity takes place. That leads to an intradermal reaction, whose diameter is measured after 48-72h post-inoculation.

The antigens contained in PPD are not specific of M. tuberculosis, which makes гnespecificity an important drawback of TST. In fact, a BCG-vaccinated individual normally responds to PPD regardless the exposition to the pathogen. Furthermore, an individual exposed to non-tuberculous mycobacteria can also generate a response against PPD. As false positive results can be obtained, although the test is considered positive when the induration is 5mm or higher, the interpretation of the results has to be done carefully done.

Another drawback of TST is its low sensitivity, especially in individuals with a compromised immunity, such as immunosuppressed patients and young children. These groups have a high risk to progress to active TB once infected.

Interferon- release assays (IGRAs)

IGRAs are in vitro blood tests designed for a decade ago to diagnose LTBI. They are used in clinical practice in some countries but they are still being studied.

Currently, two commercial tests are available: T-SPOT.TB (Oxford Immunotec, UK) based on ELISPOT and QuantiFERON-TB Gold In Tube (Cellestis, Ltd, Australia) based on ELISA.

The basis of IGRAs is the stimulation of whole blood (or isolated mononuclear cells) with specific M. tuberculosis antigens and the detection of released IFN- by sensitized T cells as a response. Depending on the method used –ELISPOT or ELISA-, what is measured is the number of T cells producing IFN- or the IFN- released amount, respectively.

The antigens used in IGRAs for stimulating the sample are ESAT-6, CFP-10 and TB7.7. They are all not found in BCG strain or in the most non-tuberculous mycobacteria (NTM), so are almost specific for M. tuberculosis, although some other mycobacteria contain also these antigens.

IGRAs present some advantages over TST, such as a higher specificity, and also a higher sensitivity in immunossuppresed individuals.

Many studies have addressed the usefulness of IGRAs in the detection of LTBI. They seem to be better than TST, in the correlation with risk factors, in calculating the positive predictive value, in avoiding false positive results due to BCG vaccine or to NTM sensitization, in the LTBI diagnosis of immunosuppressed individuals.

However, there are still some uncertainties, such as their real predictive value and the clinical significance of conversions and reversions.

Active TB Infection Diagonostics

Microbiological diagnostics

This is a summary of the procedure used in Hospital Germans Trias i Pujol for the microbiological diagnosis of tuberculosis.

- Decontamination of the sample

- Smear examination by fluorescence microscopy

- Culture in liquid and solid media

- Mycobacterium tuberculosis complex (MTBC) identification

- Non-tuberculous mycobacteria (NTM) identification

- Drug susceptibility testing for first- and second-line drugs

- GeneXpert

Mycobacterium tuberculosisis a pathogen that can cause disease -pulmonar and extrapulmonar TB- in all human tissues, therefore it can be found in all types of samples, but the most common one is the sputum sample.

Decontamination of the sampleis performed in order to a) homogenize the sample to release the possible mycobacteria present inside the cells, b) eliminate fast growing bacteria that would contaminate the culture before the mycobacteria is able to grow (division time 18-24h), and c) concentrate the mycobacteria.

For the decontamination of the sample, in Hospital Germans Trias i Pujol, we follow the Kubica N-acetyl-L-cysteine NaOH method.

We perform smear examination of auramine stained samples by fluorescence microscopy. The staining is based in mycobacteria the acid fast bacilli (AFB) property they resist decolorization by acid-alcohol due to the high lipid (mycolic acid) content in their cell walls. Fluorescence microscopy is faster and more sensitive than Ziehl-Neelsen, although less specific. In positive doubtful cases, slides may be restained with Ziehl-Neelsen to confirm and differentiate morphology.

In Hospital Germans Trias i Pujol, all the samples are cultured in liquid - BACTEC MGIT 960 - and solid media - Löwenstein-Jensen and Coletsos.

The BACTEC MGIT 960 system is a non-radiometric method. The metabolic activity of microorganisms leads to the consumption of oxygen and nutrients in the culture medium. The culture vials (modified Middlebrook 7H9) contain a fluorescent compound included in the silicone on the bottom of the tube that responds to the oxygen concentration in the culture medium. Initially, the concentration of oxygen quenches emissions from the compound, so little fluorescence is detected. Later on, microorganisms grow actively and consume oxygen, allowing the compound to emit fluorescence. The instrument automatically tests tubes continuously activating sensors and lighting the bottom of the tubes and collecting the readings, so the level of fluorescence measured by photosensors is directly proportional to the amount of oxygen consumed by microorganisms, indicating the positive cultures.

In order to differentiate between Mycobacterium tuberculosiscomplex (MTBC) and non-tuberculous mycobacteria (NTM), we use an immunochromatographic test based on the presence of MPT64 antigen.

We perform the non-tuberculous mycobacteria (NTM) identification by line probe assay (LiPA). This test is based in DNA amplification of conserved DNA regions among the mycobacteria and reverse hybridization in nitrocellulose strips.

We perform drug susceptibility testing (DST) with MGIT only for MTBC cultures. In case the isolate is resistant to first-line drugs, we perform DST for second-line drugs.

GeneXpert MTB/RIF(Cepheid, USA) is an automated device that allows the detection of MTBC in sputum samples and the resistance to rifampicin, commonly associated with multi-drug resistant tuberculosis (MDR-TB). This test is based on real-time PCR and the result can be read in 1h and 45min.

Differences in diagnostics procedures in TB Clinics, Odessa, Ukraine

There are some differences in the procedure used in the TB clinics in Odessa, Ukraine, for the microbiological diagnosis of TB. First of all, Ziehl-Neelsen smear examination is performed from direct sputum, before decontamination. The procedure of sample decontamination before inoculation of BACTEC MGIT 960 tubes is performed using BBL™ MycoPrep™ Reagent, a NALC-NaOH commercial soultion. Mycobacterium tuberculosis complex is differentiated from non-tuberculous mycobacteria by immunochromatographic test (MPT64 antigen) when grown in liquid culture and by culturing in presence of p-nitrobenzoic acid on solid medium. GeneXpert MTB/RIF analysis is 6 times cheaper in Ukraine comparing to Spain (10 euro vs 60 euro) and HIV positive subjects with suspicion of TB disease and subjects with relapsing TB are routinely tested with GeneXpert MTB/RIF (up to 20 tests/day). Finally, the DST to second-line drugs is performed in Löwenstein-Jensen solid media.

 

 

 

 

 

Tuesday the 25th.