Tuberculosis (TB) is a disease caused by the bacterium Mycobacterium tuberculosis (MTB). It ranks as the second leading cause of death from a single infectious agent, after the human immunodeficiency virus (HIV).

BURDEN OF DISEASE

Global Worldwide, 9.6 million people are estimated to have fallen ill with TB in 2014, of which 12% were HIV-positive. An estimated 1.5 million people died worldwide in 2014 due to TB. An estimated 480 000 cases of multidrug-resistant TB (MDR-TB) occurred in 2014, but only about a quarter of these

were detected and reported. Only 50% of MDR-TB cases were successfully treated in 2014. An estimated 9.7% of people with MDR-TB have extensively drug-resistant TB (XDR-TB). 105 countries have reported XDR-TB cases by 2015.

From 2016, the goal is to end the global TB epidemic by implementing the End TB Strategy, which was adopted by the World Health Assembly in May 2014. The strategy serves as a blueprint for countries to reduce the number of TB deaths by 90% by 2030 (compared with 2015 levels), cut new cases by 80% and ensure that no family is burdened with catastrophic costs due to TB disease.

South Africa South Africa had the sixth largest absolute number of incident TB cases in 2014, but in terms of incidence rate per

100 000 population we were second (after Lesotho). In 2014, an estimated 61% of TB patients were also HIV-positive, of which 79% received antiretroviral therapy. In 2013 78% of all new and relapsed TB cases in South Africa were treated successfully. In 2014 the mortality rate due to TB was 44 per 100 000 population in patients who were not co-infected with HIV,

but the mortality rate was significantly higher amongst HIV co-infected people (134 per 100 000 people).

CLINICAL PRESENTATIONTB disease should be suspected in persons who have the following symptoms: Unexplained weight loss Loss of appetite Night sweats Fever Fatigue

Symptoms of pulmonary TB may include: Coughing for > 3 weeks Haemoptysis (coughing up blood) Chest pain

If TB disease occurs outside the lungs (extrapulmonary), symptoms will depend on the area affected.

TB/HIV Co-infectionHIV-infected individuals have a 5% - 10% annual risk of developing TB disease vs. a 10% lifetime risk for HIV-uninfected individuals. The risk of developing TB disease increases as the CD4+ count drops.

DIAGNOSISThere should be a high index of suspicion for TB disease, especially in the HIV-positive population. Once the diagnosis is considered based on the history and clinical examination, diagnosis is confirmed by detection of the organism in secretions or tissue.

Compiled by Dr K. Pillay

TB DIAGNOSTICS1 Quarter 2016st

TBEND

WORLD TB DAY | MARCH 24

0861 LANCET(526238)

Radiological examinations are useful, but may be inconclusive especially in HIV-infected individuals with a low CD4+ count. In some circumstances, establishing a definitive laboratory diagnosis of TB may not be possible. A specific bacteriologic confirmation is never established in 15% - 20% of patients with a clinical diagnosis of TB. In such cases, a presumptive clinical diagnosis is sufficient for initiating therapy.If clinical suspicion for TB is high, initiation of empiric therapy pending laboratory diagnostic confirmation is appropriate.

Clinical specimensSite specific tissue or fluid is recommended for testing e.g. sputum for pulmonary TB, CSF for TB meningitis. Blood is not an appropriate specimen except from bone marrow aspirates on which cultures may be performed.

SPUTUMFor the diagnosis of pulmonary TB, testing on three sputum specimens is recommended. These should be obtained at least 8 - 24 hours apart and should include at least one early morning specimen. Sputum may be obtained spontaneously (by coughing) or may be induced. Nasopharyngeal secretions and saliva are not sputum and are inadequate for testing.Sputum may be induced by inhalation of aerosolized hypertonic saline generated by a nebulizer. This should be performed by trained personnel. Such specimens should be clearly labelled �induced sputum�. Induced sputum is comparable to bronchoalveolar lavage specimens, while being safer and less costly.

GASTRIC SECRETIONSGastric aspiration can be useful in children who cannot produce sputum (even with induction).

TISSUE BIOPSYTissue biopsy may establish a definitive diagnosis of tuberculosis when non-invasive techniques are not diagnostic. Biopsy specimens from the lung, bone marrow, pericardium, lymph nodes, bones, joints, bowel, liver, brain, or other tissues allow for both histopathologic examination and microbiological studies. Biopsy specimens should be collected with and without fixative; specimens without fixative are required for culture.

Laboratory testingSTAINED SMEARSThe detection of acid-fast bacilli (AFB) on microscopic examination of stained smears is the most rapid and inexpensive TB diagnostic tool, however it lacks sensitivity. The sensitivity and positive predictive value of AFB smear microscopy are approximately 45% - 80% and 50% - 80%, respectively. According to the WHO Global Tuberculosis Report, only 40% of all cases of TB diagnosed in South Africa in 2011 were smear-positive. Sensitivity increases when multiple specimens are obtained, but the sensitivity of stained smears is lower in patients with a low organism burden, such as children with primary TB and HIV-positive patients with paucibacillary disease.Acid-fast bacteria visualized on a slide may represent M. tuberculosis or non-tuberculous mycobacteria, so species identification requires molecular testing and/or culture.In the setting of a high clinical suspicion for TB, initiation of empiric therapy based on a positive AFB stain is appropriate. A positive AFB stain also helps to identify those individuals who are more infectious. AFB smears quantitate the number of organisms seen and should decrease sequentially in patients who are responding to treatment. This data may be used together with clinical and radiographic information to monitor response to treatment.

Fig. 1: Acid-fast bacilli

CULTUREAll clinical specimens suspected of containing mycobacteria should be cultured. Culture can detect as few as 10 bacteria/mL. Culture is required for drug susceptibility testing and can also be used to identify non-tuberculous mycobacteria. The sensitivity and specificity of sputum culture are about 80% and 98%, respectively. The disadvantage of culture is that it can take 2 - 6 weeks to obtain a final result.Growth in liquid media is faster (1 - 3 weeks) than growth on solid media. Lancet Laboratories uses an automated liquid broth culture system for the detection of mycobacteria, namely MGIT [mycobacterial growth indicator tubes].Once growth is detected, species identification is performed using molecular methods.Drug susceptibility to the first line anti-TB drugs can also be performed on the positive culture, but this takes an additional 2 - 3 weeks for a final result.False-negative cultures may occur when bacilli are killed during decontamination or if an inadequate specimen is submitted. A contaminated culture means that the patient's own normal bacterial flora was present in the sputum and overgrew in the MGIT tube, so even if MTB was present, it could not be detected. Repeat decontamination can further destroy MTB thus decreasing the yield of bacilli.

MOLECULAR TESTSDetection of mycobacterial DNA can be used for rapid detection of organisms belonging to the M. tuberculosis complex, allowing timely initiation of appropriate treatment as well as infection control measures. Molecular testing has excellent positive predictive value in the setting of AFB smear positive specimens for distinguishing tuberculous from non-tuberculous mycobacteria (> 95%), and it can rapidly establish the presence of tuberculosis in 50% - 80% of AFB smear negative specimens (which would eventually be culture-positive). A negative molecular test result is not sufficient to exclude TB and negative results should be confirmed with culture. Molecular test results must be interpreted in the context of clinical and epidemiologic factors. In addition, molecular testing can detect DNA from dead and live organisms, so testing can remain positive for some time after appropriate therapy.Molecular testing can be performed on specimens other than respiratory secretions, although this is an �off-label� application in many instances.

Xpert MTB/RIF assay The Xpert MTB/RIF assay is a molecular test that can simultaneously identify M. tuberculosis and rifampicin resistance. The test correctly identifies 98% of patients with smear positive TB and 72% smear negative/culture positive TB. The accuracy for identification of rifampicin resistance is 98%. The Xpert MTB/RIF is approved for testing on smear-positive and smear-negative sputum samples from patients with suspected pulmonary TB who have received fewer than three days of treatment. The test may also be used for samples other than sputum in �off label� applications, if validated appropriately by the laboratory.

Line probe assay [HAIN]This PCR-based assay is able to detect mycobacterial DNA in smear-positive sputum and cultured isolates. It is able to test for both isoniazid and rifampicin susceptibility. It can also be used for species identification of mycobacteria other than TB (MOTT). This assay requires more specialised equipment, cannot be used directly on smear-negative sputum and has a longer turnaround time than the Xpert MTB/Rif assay; for these reasons it is mainly used for testing on cultured isolates.

LAM ANTIGEN DETECTIONLipoarabinomannan is a glycoprotein found in the outer cell wall of mycobacteria. LAM is released from metabolically active or degrading bacterial cells during TB infection. LAM is filtered by the kidneys into the urine. A rapid antigen detection assay is available for the detection of LAM in urine. The sensitivity is optimal in HIV-positive patients with low CD4+ counts and the sensitivity increases as the CD4+ count drops.

Fig.2: The biosafety level 3 TB lab at Lancet Laboratories

LAM testing can be used for the detection of TB in HIV-positive patients with CD4+ counts < 200 cells/�L, especially if disseminated TB is suspected.It is important to note that: A negative LAM does not exclude TB A positive LAM does not distinguish between TB and infection with other mycobacteria e.g. Mycobacterium avium

TUBERCULIN SKIN TEST (TST)An intradermal injection of 0.1mL tuberculin purified protein derivative (PPD) on the inner surface of the forearm can be used to determine if someone has been infected with MTB. It is most frequently used in children. It is not useful for the diagnosis of active disease in adults. Testing requires two visits � one for the injection and a follow up visit 48 - 72 hours later for the test to be read. Induration at the injection site is measured. HIV infection can lead to anergy and false-negative results.

INTEFERON GAMMA REALEASE ASSAY (QuantiFERON - TB Gold Test)The patient's blood is incubated with TB antigens. The release of gamma interferon from white blood cells in response to the TB antigens is measured. This can be used to detect the presence of latent MTB infection. It is not useful for the diagnosis of active disease in adults. HIV infection may lead to false-negative IGRA results, but a false-negative is less likely to occur compared with the TST.

ROLE OF SEROLOGYThere is no role for serologic testing in the diagnosis of TB. In 2011, the World Health Organization (WHO) issued a strong negative recommendation against their use based on a meta-analysis of 92 studies concluding that the use of commercial serological tests is supported only by data of very low quality.

References:http://www.who.int/tb/publications/global_report/en/http://www.info.gov.za/view/DownloadFileAction?id=72544,http://www.uptodate.com/contents/diagnosis-of-pulmonary-tuberculosis-in-hiv-negative- http://familymedicine.ukzn.ac.za/Libraries/Guidelines_Protocols/TB_Guidelines_2009.sflb.ash

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