INTRODUCTION 1.8 millions of death because of tuberculosis. Particularly

INTRODUCTION      Tuberculosis (TB)  is one of world’sproblem related to morbidity and mortality.1 In 2015, over 10.4 millions of TB cases in the world and 1.8 millions ofdeath because of tuberculosis. Particularlyin Indonesia, tuberculosis has infected 1,02 million people and became one oftop 5 countries in the world infected by tuberculosis.2 In 2013, the prevalence of tuberculosis is 0.

4 %.3From top 10 worldwide causes ofdeath in 2015, tuberculosis seed in number 9 and number 5 for low-incomecountries such as Angola, Bangladesh, Brazil, Ethiopia, Congo, Indonesia,Nigeria, etc. Those top causes of death are ischaemic heart disease and strokeas the world’s biggest killers, lower respiratory infections as the most deadlycommunicable disease, chronic obstructive pulmonary, trachea and lung cancer,diabetes mellitus, Alzheimer, diarrhoeal, tuberculosis, and road injury.

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2 In Indonesia, tuberculosis has a significant impact on economic aspect as75% of TB survivor is the productive group (15-50 years old).4The number of mortality remainshigh as the diagnosis is delayed and uncontrolled transmission.5 The major challenge in conventional methods for TB diagnosis is the poorsensitivity to detect Mycobacterium (20-80%).5–7 The diagnosis of extrapulmonary TB (EPTB) faces some problems due to thelimited number of bacilli in the specimen and the difficulty in obtaining thespecimen from the site or organ infected.8 Culture is the reference standard to diagnose TB; it needs 2-8 weeks toyield the Mycobacterium.9,10 Rapid detection of TB is needed to start antituberculosis treatment so itcan prevent its further transmission.1,7Other methods to detecttuberculosis are the serologic test (tuberculin skin test/TST),interferon-gamma release assays (IGRAs), and nucleic acid amplification test(NAAT).

TST is a delayed type hypersensitivity test that will occur in infectedperson. It has several limitations such as antigenic cross-reactivity, lowsensitivity, low specificity, and false positive result. IGRAs used thedetection of interferon-gamma (IFN-?). In comparisonto TST, it is useful to identify infected a person with the immunocompromisedcondition and inflammatory disease.

11GeneXpert MTB/RIF (Cepheid,Sunnyvale USA) is a molecular assay to detect Mycobacterium tuberculosis that gives more advantages thanconventional methods.2 GeneXpert MTB/RIF assay uses quantitative real-timepolymerase chain reaction (qRT-PCR) test to detect both M. tuberculosis and rifampicin resistance within 2 hours.12–14 This review will discuss the basic concept of tuberculosis, differenttypes of diagnostic methods, and specimens as the samples for TB diagnosis.TUBERCULOSIS: THEMICROBE AND PATHOGENESISTuberculosis is an infectious disease caused by Mycobacterium tuberculosis complex (MTBC).15 Besides Mycobacterium tuberculosis, it consists other species such as M.

africanum, M. microti, andM. bovis.16 The genus of Mycobacterium iscategorized as acid-fast bacilli (AFB) due to its resistance to acid-alcoholdecolorization. 4TB infection is initiated by aerosol inhalationthat contains M.tuberculosis.

17 After inhalation, droplet nucleus is deposited into bronchialtree and attached to bronchiole or alveoli.18,19 Initial TB infection is characterized by innate immune response involvinginflammatory cells. The adaptive immune response ischaracterized by the dissemination of the bacteria in lymph nodes where antigenpresentation in dendritic cells leading to T cell activation and expansion tothe lungs. The insertion of T cell, B cell, macrophage, and leucocyte triggers a formation of granuloma that contains  M. tuberculosis.17 Mycobacterium tuberculosis is mostly digested by alveolarmacrophages, but some of them multiply intracellularly and are released afterthe macrophages dead.

  If the macrophagesare still alive, the bacteria will spread into the lymphatic channel orbloodstream to the tissues/organs, and develop extrapulmonary TB (EPTB).18TB transmission can occur when an infected person spreadsdroplet nuclei into the air. The main symptoms of TB in adults and children area persistent cough, lethargy weight loss, fever, and night sweats.20 TB transmission is determined by some factors. First, a susceptibilitythat is shown by positive AFB smear or radiographic findings.

These suggestivefindings are hylus lymphadenitis, segmental/lobar consolidation, pleuraleffusion, miliar, atelectasis, infiltrate calcification, and tuberculoma.Second, is contact history with TB survivor and the contact duration.  Third, is space of exposure where the small,enclosed spaces, and inadequate ventilation ease the transmission. Fourth, isthe infectivity of bacteria’s strain.19,21DIAGNOSIS: METHODSAND SAMPLESA complete medical evaluation of TB includes medical history, physical examination, and furtherexamination to find M.

tuberculosisinfection.2 In pulmonary TB, the etiological diagnosis is basedon the results of microscopic, culture, and rapid tests. If the results arenegative, the clinical diagnosis and further tests should be performed. In extrapulmonary TB, the diagnosis is determined through clinicalexamination as well as bacteriological and/or histopathology findings from thesite or organ affected.4There are some diagnostic methods of tuberculosisinfection. The first and the most common method is AFB staining withcarbolfuchsin (ZN and Kinyoun-Gabbet KG methods) and fluorochrome(auramine-rhodamine). The second method is the cultivation of specimens using aspecific grow the bacilli.

The third is serological assay with tuberculin skin test (TST) or Mantoux test. The fourth isInterferon Gamma Release Assay (IGRA), and the last is nucleic acidamplification test (NAAT)  with GeneXpert MTB/RIF assay (GeneXpert).11The results of acid-fast stainingare interpreted according to the classification set by the World Health Organization(WHO) criteria. This diagnostic method is inexpensive and rapid but has poorsensitivity and depends on the skill of the staff performing the test.

14,20 If the result of AFB staining is negative but theclinical suspicion is high, culture should be performed to identify the growthof M. tuberculosis.11,22Culture is a gold standard to diagnose M.tuberculosis bacilliinfection.11,19 Thespecimen used to grow the bacteria is taken from the site or organ wheretuberculosis infection occurs, such as sputum, urine, cerebrospinalfluid (CSF), pleural fluid, pus, and tissue biopsy. Sputum iscollected through various ways; for example, spontaneous coughing, coughing induction using salineinhalation, bronchoscopy, and gastric aspiration.20Culture becomes an important test of tuberculosis as it can also analyzedrugs susceptibility and increase the detection of smear-negative AFB. It canbe performed in solid or liquid media such as Lowenstein Jensen (LJ),Middlebrook 7H11, BACTEC MGIT 960 System, and Bactec 9000MB, etc.

6 The specimen to becultured is processedthroughdigestion, decontamination with N-acetyl-l-cysteine and sodium hydroxide (NALC/NaOH) and centrifugation 3500g for 15 minutes. Then, the specimen isresuspended in 1 mL buffer phosphate sterile (pH = 6.8). Theprocessed specimen is then being inoculated into a solid medium (LJ) with avolume of 0.

2 mL and liquid media such as MB/BacT (bioMe´rieux, Marcyl’Etoile, France) and Bactec MGIT 960 with a volume of 0.5 mL. Lastly, thespecimen is incubated at 37 0C for 6 weeks.6,23The culture of M. tuberculosis isable to detect 10-102 organism/mL specimen and has highersensitivity than AFB staining. However, the growth M. tuberculosis is very slow; thus, culture requires 8 weeks ofincubation.

1 Inaddition, it also needs a proper laboratory infrastructure and skilled staff.7,22,24Tuberculin skin test uses a substance called tuberculin which is 200 purifiedprotein derivate (PPD) that can induce a delayed-type hypersensitivity afterbeing administered under the skin. A positive in vivo reaction showed the diameter of induration about 5 mm in the immunocompromised patientand 10 mm inimmunocompetent children and adults who never received vaccination and 15 mm inthose who have been vaccinated. The limitation of this test is that it has poorsensitivity, specificity, and there is a possibility of cross-reaction withother antigen(s).11,19 Anotherlimitation is that it can cause false positive result in a person who has beenvaccinated with Bacilli Calmette-Guerin (BCG).11Interferon-gammarelease assay IGRA is a method of diagnosis performed by measuring the immuneresponse of interferon-gamma (IFN-?) to TB antigens including early secretory antigenictarget-6 (ESAT-6), cultures filtered protein-10 (CFP-10), and TB7.7. Thoseantigens were encoded by the gene in a region of difference-1 (RD1) of M.

tuberculosis complex. Becausethis method detects the host immune response to TB antigens located in thespecific regions of the bacterial gene, the method has higher sensitivity and specificitythanTST.11,19The IGRA method is based on enzyme-linked immunosorbent assay (ELISA) andELISpot assay. An example of the ELISA-based kit is QuantiFERON-TB Gold In-Tube(QFT-GIT) (Cellestis Ltd., Carnegie, Australia) whereas T-SPOT.

TBTM test(Oxford Immunotec Inc., Abingdon, UK) is based on the ELISpot assay. Thespecificity of IGRA is higher than TST so IGRA is a very useful method for thescreening of tuberculosis, TB/HIV coinfection, and TB in immunocompromisedpatients. The limitation of IGRA is due to the availability of the mononucleatedcells in peripheral blood that can affect the result and give false positive ornegative.11Thelast method for TB detection is molecular detection. The method has a rapidturn-around time so it can overcome the slow-growing bacteria in cultivation.

11 A novel and advanced techniquefor TB molecular detection is GeneXpert MTB/RIF assay. The assay is a qRT-PCRbased molecular test that detects the presence of M. tuberculosis within less than 2 hours of analysis.11,25 This assay was endorsed by WHOin 2010 as the rapid diagnosis test to detect TB and rifampicin resistance.2,26 GeneXpert MTB/RIF is anautomatic cartridge-based assay that has many benefits. First, the closedamplification system can reduce the potency of cross-reaction. Second, theGeneXpert assay can give information about rifampicin resistance coded in rpoBgene. The rpoB gene is responsible for 96% of rifampicin resistance cases in M.

tuberculosis infection. Therifampicin resistance is a key predictor for multidrug-resistanttuberculosis  (MDR-TB) as most of thebacteria resistant to rifampicin is also resistant to isoniazid.12GeneXpert MTB/RIF processesthe specimens and analyzes them automatically. The platform consists of two components, i.e.: (i) cartridge that contains sample reagent (SR), PCR buffer and qRT-PCR lipophilic reagent, and (ii) GeneXpert instrument thatcontrols the liquid inside the cartridge and performs a qRT-PCR analysis. The GenXpert instrument amplifiesthe sequence of rpoB gene that specific to the nucleic acid of M. tuberculosis.

The primer sequence of M. tuberculosis and rpoB-specific molecular beacons are modified to run heminested PCR so that does notreport non-tuberculous mycobacterium (NTM) and to maximize mutation detection as thepredictor of MDR-TB.12,24M. tuberculosis is detected using five overlapping molecular probes (A–E) in region 81-bp rpoB. It isidentified with at least two of the five probes shows positive signals with cycle threshold of 38 cycles orless.A cycle threshold (CT) is anumber of cycles needed to obtain the positive result.

The range of CT isclassified into high, <16; medium, 16–22; low, 22–28; very low, >28.13The sample reagent containssodium hydroxide (NaOH) and isopropanol with the ratio of 3:1 is added to 0,5mL  sample.7,26 The sample reagent is used to dilute the sample, toreduce the infectiousness of the sample, and to inactivate PCR inhibitors.6 The container consisting specimen is agitated twicewithin 15 minutes and incubated at room temperature pressure (r.t.

p).22,26 A volume of 2 mL mixture is inserted into thecartridge and then the PCR assay is run by GeneXpert automatically to detect M. tuberculosis and rifampicinresistance (RIF).6 The result is ready within less than 2 hours.

26 In pulmonary TB, the assay can be done using eitherraw sputum or pellet sputum.7,12 During the GeneXpert assay, there is a chance to obtain false negative orpositive results. False positive results, often caused by false amplificationtarget,  whereas false negative results oftenrelated to the liquid problem or sample inhibitor.12 The errorof sample processing is reduced by using Bacillusglobigiispore asthe internal control. The spore is inserted into the cartridge to ensure thelysis M. tuberculosis and to detect PCR inhibitors.28 The GeneXpert instrument requires astable electricity, appropriate temperature (<30 0C), and routinecalibration of the cartridge module.5,7 The time difference ofdiagnosis methods to detect M.

tuberculosis can be seen in Table 1. Table 1. Time Difference of Laboratory Procedure to Detect the Presence of M. tuberculosis Test Time required I.       Nucleic acid amplification test, detection (NAAT-TB) II.      Nucleic acid amplification test, resistance markers (NAAT-R) III.     Acid-fast bacilli microscopy IV.

    Growth detection Liquid Solid V.     Identification of Mycobacterium  tuberculosis complex by DNA probe/HPLC VI.    First-line drug susceptibility testing (liquid medium) VII.   Second-line and novel compound drug susceptibility testing Liquid (broth-based) medium Solid (agar/egg-based) medium 1 d 1-2 d 1 d   Up to 6-8 wk (average 10-14 d) (average 3-4 wk) 1 da 1-2 wka 1-2 wka 3-4 wka  Abbreviation:HPLC, high-performance liquidchromatography. aafter detection of growthAdopted fromDiagnosis of TB in Adults and Children, IDSA Guideline.

19 SAMPLESTuberculosis can be divided into pulmonary and extrapulmonary. It isestimated that 15-20% of extrapulmonary TB affects the lymph nodes, meninges,kidney, spine, and bone. Extrapulmonary TB (EPTB)  is an atypical clinical presentation thatoften stimulated by neoplasia or inflammation.29 For diagnosing EPTB, the specimens can beobtained from pleural fluid, aspirate or tissues from lymph node, or CSF.30 Either pulmonary or extrapulmonary TB, there are some specimens that can besubjected to GeneXpert assay.

It is important to note, however, that specimensfrom the outside of respiratory system contain a low number of bacilli. Thespecimens microbiology diagnostics should be collected from sterile locations.SR is added to ratio 2:1 except for CSF.

The collected sample is then incubatedat room temperature for 15 minutes. Following incubation, the sample is inserted into the cartridge andtested using GeneXpert.29 Lymphadenitis TB is the most frequent EPTB form. The sample can be obtainedfrom the tissue or aspirated using fine needle biopsy from the lymph node. Theuse of fresh sample will give higher sensitivity than the frozen sample.30 In pleural TB, the sample is taken frompleural fluid whereas meningitis TB can be diagnosed by using CSF sample. Areview by Denkinger et al. (2014) shows that the use of GeneXpert has high sensitivity to diagnose lymphadenitis TB, medium sensitivity in the diagnosis of meningitisTB and low sensitivity in pleural TB.

8Another research that used various samples to diagnoseEPTB shows different sensitivities. The sensitivity of the assay when usingtissue biopsy, fine needle aspirate, gastric aspirate, pus, CSF,  and urine is 75%, 88.3%, 78.7%, 87.3%, 85.7%, and 87.

5% respectively. Lower sensitivity valuesare obtained when using samples from pleural fluid (44.4%) andother body fluids such as pericardium, peritoneum and synovial fluid (50%).8Sputum is the most frequent sample used to diagnose pulmonary TB. Sputumsample can be differentiated into raw and pellet sputum. Gastric aspirate canbe used as GeneXpert sample from old patients who cannot expel sputumeffectively.

31 Another sample that can be used is a fecalspecimen. The required volume of specimen is 2 cm3 and then 10 mLadded with distilled water and then it is homogeneously using vortex.31The mixture of the specimen and distilled water is incubated at roomtemperature for 15 minutes and then being centrifuged 3000 x g for 20 minutes.Following this, the sediment is decontaminated using 10 mL of NALC/NaOH 3% for15 minutes at room temperature. The next step, the sample is mixed with 40 mLof phosphate buffer with pH of 6,8 and then centrifuged 3000 × g for 20minutes. The sediment is then resuspended in 3 mL of phosphate buffer and theninoculated in Ogawa medium and MGIT.

The remaining sample can be stored at ?80 °C for 1-3months.31 Raw sputum andpellet sputum can be used to examine using GeneXpert as the recommendation fromCepheid. The pellet sputum is a raw sputum that is processed by liquefaction, decontamination usingNALC/NaOH and centrifugation.

6The raw sputum can be obtained either spontaneously expectorated bythe patients or after sputum induction. The required volume of pellet sputum is0.5 mL and raw sputum is 1 mL.28The types of sputum influence its storage and transport. Pellet sputum canbe stored at 2–8 °C for up to 7 days whileraw sputum can be stored at the maximum temperature of  35 °C for 3 days andthen moved into2–8 °C for the next 7 days.

The sample reagent is used to liquefyraw and pellet sputum with the ratio of 2:1 and 3:1, respectively.28 Previous research reported that thesensitivity of GeneXpert using raw or pellet sputum is similar.6CONCLUSIONTuberculosisas one of the major global problems can be diagnosed using several methods.Culture as the gold standard requires a long time to grow the bacteria. Themicroscopic examination and tuberculin skin test have poor sensitivity.

Interferon-gamma release assays with a better specificity than TST is affectedby mononucleated cells. GeneXpert works rapidly and automatically so it canincrease TB detection using various kinds of specimens regarding the site ororgan.