ESTIMACIÓN DE RECURSOS

Definitive diagnosis can only be attained based on bacteriological investigations.

According to the WHO (2014a), among pulmonary TB cases, 58% were bacteriologically confirmed (as opposed to clinically diagnosed) in 2013. The remaining 42% of patients who were not bacteriologically confirmed could result in a false diagnosis with individual’s enrolment on TB treatment when not necessary. Furthermore, a low rate of laboratory confirmation reflects an under-diagnosis of true TB cases and contributes in part to the continuing global gap between notified and estimated incident TB cases (5.7 and 9.0 million in 2013, respectively). Also, detection of TB without investigating for drug resistance can lead to ineffective treatment, further development and spread of drug-resistant strains and additional suffering and costs for patients.

All forms of bacteriological TB diagnoses occur by using a variety of bacteriological investigation methods (NDoH, 2014). The diagnostic tests available to confirm

bacteriological TB include: (i) Smear Microscopy, (ii) Culture Methods, and (iii) Molecular Methods [Polymerase chain reaction (PCR) based assays: Line probe assay and Xpert MTB/RIF].

Microscopy and culture are still the basis of TB diagnostics. Sputum smear microscopy is the most common method for diagnosing TB worldwide (Desikan, 2013;

NDoH, 2014). Sputum smear microscopy entails the examination and observation of the bacilli in sputum samples under a microscope (Desikan, 2013; NDoH, 2014), using either one of two staining methods, namely the Ziehl-Neelsen staining method (NDoH, 2014, Truffot-Pernot & Cambau, 1994) or fluorescent auramine staining method (NDoH, 2014). The staining procedure depends on the ability of mycobacteria to retain these dyes when treated with acid and alcohol solutions (NDoH, 2014).

The use of sputum smear microscopy is controversial. Sputum smear microscopy is fast, inexpensive and requires minimal bio-safety standards (Desikan, 2013; Steingart et al., 2006; WHO, 2014b). However, it is not a sensitive test, particularly in children and people living with HIV as it provides no information on the viability and drug susceptibility of the bacilli. The viability and drug susceptibility were observed in a study published in

Stellenbosch, South Africa, where the objective was to assess the use of a sputum register to evaluate the TB diagnostic process and the initiation of TB treatment. TB patients were classified as patients with two positive smears. Over the entire diagnostic process, up to 5%

of TB cases were missed (Botha et al., 2008). The sensitivity is grossly compromised when the bacterial load is less than 10,000 organisms/ml per sputum sample. It also cannot distinguish between MTC and non-TB mycobacteria (Desikan, 2013; WHO, 2014b).

Culture methods, in comparison, are more sensitive than smear microscopy as they detect a higher proportion of cases among patients with symptoms (David, Katalinić-Janković, Fattorini & Cirillo, 2016; Desikan, 2013). It allows for the detection of very low numbers of bacilli (approximately 10 bacilli/ml of sputum compared with at least 5000 bacilli/ml of sputum for microscopy) (David et al., 2016). The use of culture tests increases the number of TB cases found by 30–50% (David et al., 2016). Culture tests diagnose

extrapulmonary TB, distinguish TB from non-TB mycobacteria and detect treatment failures.

Culture testing is also critical for monitoring patients’ response to treatment for DR-TB.

Molecular methods have been the recent breakthroughs in TB diagnostics in the past decade, to diagnose TB and DR-TB (WHO, 2014b). In 2010, the WHO endorsed

Xpert®MTB/Rif (Cephaid, Sunnyvale, USA), a PCR-based diagnostic tool (Günther, 2014).

Molecular biology is now becoming more critical in the diagnosis of mycobacteria. It supports culture either by serving as a rapid direct test on specimens or by enabling a rapid and unequivocal species differentiation from culture material. Nucleic-acid-based methods

have primarily displaced the classical methods (Hillemann, Miychell, Drobniewski, 2016). In countries with more developed laboratory capacity, cases of TB are also diagnosed via

culture methods (the current reference standard) (WHO, 2014b). Molecular genetic tests offer considerable time advantages in the identification of mycobacteria, enabling a more rapid initiation of resistance tests and specific treatment. Molecular methods are useful tools for the detection and differentiation of mycobacteria from cultures and can have a high specificity and sensitivity. It should be stated, however, that they cannot/should not replace the currently endorsed standard methods of detecting mycobacteria and determining drug-susceptibility patterns. Instead, their use should support the diagnostic work-up. Confirmation of the test results should always be through the use of the standard methods (Hilleman et al., 2016).

In South Africa, there are two PCR technologies available which provide different information that is helpful in the management of TB and DR-TB. They include the

Xpert®MTB/Rif test using the Gene Xpert (GXP) instrument, which is useful for rapidly diagnosing TB as well as it allows rapid screening for Rifampicin resistance. Results can be available within two hours in the laboratory but only become available within 48 hours in health facilities (NDoH, 2014). In clinical evaluation studies, its sensitivity approaches 100%

in smear-positive pulmonary TB patients and 57–83% in patients with smear-negative pulmonary TB (Pontali et al., 2013).

As per the South African guidelines when diagnosing DR-TB rapid testing using Xpert^®MTB/RIF, confirmatory drug susceptibility testing for patients with RR-TB, rapid tracing and linkage to treatment and rapid tracing and evaluation of contacts is suggested (NDoH, 2014). Gene Xpert is the method most commonly used in South Africa.

Line probe assay is the second molecular test that can be performed on all respiratory specimens and other specimens where the detection of rifampicin resistance in MTB is the primary purpose of the investigation (Hilleman et al., 2016). This is useful for drug resistance

confirmation and detects resistance to both Rifampicin and Isoniazid. Line probe assays is specific for MTB complex and can differentiate MTB from other mycobacteria. Line probe assays can only be performed on smear positive or culture positive sputum specimen (NDoH, 2014).

Where molecular tests, such as the GeneXpert are available, culture may still be required for HIV positive patients where TB is suspected yet have a negative GeneXpert test (NDoH, 2014) and RR-TB cases, where testing for another drug resistance is necessary (NDoH, 2014).

There are a few other bacteriological tests that can be used in conjunction with the methods mentioned above but should not be used in isolation as a diagnostic test. These tests include: (i) Interferon gamma Release Assays (blood tests that detect MTB infection but cannot distinguish latent TB from active TB), (ii) blood culture (may be used to detect MTB and other species of mycobacteria in HIV-infected patients; especially those with low CD4 count), (iii) TB LAM (detects lipoarabinomannan antigens in urine - further studies are however required to determine the role of this test), (iv) histological examination (can be conducted on a tissue specimen, but this is not considered to be bacteriological confirmation of disease) and (v) the Tuberculin skin test (limited value in clinical work as the test shows hypersensitivity to proteins of the TB bacillus, as a result, either of infection with M.

tuberculosis or induced by Bacille Calmette-Guérin vaccination) (NDoH, 2014).