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INTRODUCTION 1.8 millions of death because of tuberculosis. Particularly

INTRODUCTION

      Tuberculosis (TB)  is one of world’s
problem related to morbidity and mortality.1 In 2015, over 10.4 millions of TB cases in the world and 1.8 millions of
death because of tuberculosis. Particularly
in Indonesia, tuberculosis has infected 1,02 million people and became one of
top 5 countries in the world infected by tuberculosis.2 In 2013, the prevalence of tuberculosis is 0.4 %.3

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From top 10 worldwide causes of
death in 2015, tuberculosis seed in number 9 and number 5 for low-income
countries such as Angola, Bangladesh, Brazil, Ethiopia, Congo, Indonesia,
Nigeria, etc. Those top causes of death are ischaemic heart disease and stroke
as the world’s biggest killers, lower respiratory infections as the most deadly
communicable disease, chronic obstructive pulmonary, trachea and lung cancer,
diabetes mellitus, Alzheimer, diarrhoeal, tuberculosis, and road injury.2 In Indonesia, tuberculosis has a significant impact on economic aspect as
75% of TB survivor is the productive group (15-50 years old).4

The number of mortality remains
high as the diagnosis is delayed and uncontrolled transmission.5 The major challenge in conventional methods for TB diagnosis is the poor
sensitivity to detect Mycobacterium (20-80%).5–7 The diagnosis of extrapulmonary TB (EPTB) faces some problems due to the
limited number of bacilli in the specimen and the difficulty in obtaining the
specimen from the site or organ infected.8 Culture is the reference standard to diagnose TB; it needs 2-8 weeks to
yield the Mycobacterium.9,10 Rapid detection of TB is needed to start antituberculosis treatment so it
can prevent its further transmission.1,7

Other methods to detect
tuberculosis 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 infected
person. It has several limitations such as antigenic cross-reactivity, low
sensitivity, low specificity, and false positive result. IGRAs used the
detection of interferon-gamma (IFN-?). In comparison
to TST, it is useful to identify infected a person with the immunocompromised
condition and inflammatory disease.11

GeneXpert MTB/RIF (Cepheid,
Sunnyvale USA) is a molecular assay to detect Mycobacterium tuberculosis that gives more advantages than
conventional methods.2 GeneXpert MTB/RIF assay uses quantitative real-time
polymerase 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, different
types of diagnostic methods, and specimens as the samples for TB diagnosis.

TUBERCULOSIS: THE
MICROBE AND PATHOGENESIS

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis complex (MTBC).15 Besides Mycobacterium tuberculosis, it consists other species such as M. africanum, M. microti, and
M. bovis.16 The genus of Mycobacterium is
categorized as acid-fast bacilli (AFB) due to its resistance to acid-alcohol
decolorization. 4

TB infection is initiated by aerosol inhalation
that contains M.
tuberculosis.17 After inhalation, droplet nucleus is deposited into bronchial
tree and attached to bronchiole or alveoli.18,19 Initial TB infection is characterized by innate immune response involving
inflammatory cells. The adaptive immune response is
characterized by the dissemination of the bacteria in lymph nodes where antigen
presentation in dendritic cells leading to T cell activation and expansion to
the 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 alveolar
macrophages, but some of them multiply intracellularly and are released after
the macrophages dead.  If the macrophages
are still alive, the bacteria will spread into the lymphatic channel or
bloodstream to the tissues/organs, and develop extrapulmonary TB (EPTB).18

TB transmission can occur when an infected person spreads
droplet nuclei into the air. The main symptoms of TB in adults and children are
a persistent cough, lethargy weight loss, fever, and night sweats.20 TB transmission is determined by some factors. First, a susceptibility
that is shown by positive AFB smear or radiographic findings. These suggestive
findings are hylus lymphadenitis, segmental/lobar consolidation, pleural
effusion, 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, is
the infectivity of bacteria’s strain.19,21

DIAGNOSIS: METHODS
AND SAMPLES

A complete medical evaluation of TB includes medical history, physical examination, and further
examination to find M. tuberculosis
infection.2 In pulmonary TB, the etiological diagnosis is based
on the results of microscopic, culture, and rapid tests. If the results are
negative, the clinical diagnosis and further tests should be performed. In extrapulmonary TB, the diagnosis is determined through clinical
examination as well as bacteriological and/or histopathology findings from the
site or organ affected.4

There are some diagnostic methods of tuberculosis
infection. The first and the most common method is AFB staining with
carbolfuchsin (ZN and Kinyoun-Gabbet KG methods) and fluorochrome
(auramine-rhodamine). The second method is the cultivation of specimens using a
specific medium.to grow the bacilli. The third is serological assay with tuberculin skin test (TST) or Mantoux test. The fourth is
Interferon Gamma Release Assay (IGRA), and the last is nucleic acid
amplification test (NAAT)  with GeneXpert MTB/RIF assay (GeneXpert).11

The results of acid-fast staining
are interpreted according to the classification set by the World Health Organization
(WHO) criteria. This diagnostic method is inexpensive and rapid but has poor
sensitivity and depends on the skill of the staff performing the test.14,20 If the result of AFB staining is negative but the
clinical suspicion is high, culture should be performed to identify the growth
of M. tuberculosis.11,22

Culture is a gold standard to diagnose M.
tuberculosis bacilli
infection.11,19 The
specimen used to grow the bacteria is taken from the site or organ where
tuberculosis infection occurs, such as sputum, urine, cerebrospinal
fluid (CSF), pleural fluid, pus, and tissue biopsy. Sputum is
collected through various ways; for example, spontaneous coughing, coughing induction using saline
inhalation, bronchoscopy, and gastric aspiration.20

Culture becomes an important test of tuberculosis as it can also analyze
drugs susceptibility and increase the detection of smear-negative AFB. It can
be 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 be
cultured is processed
through
digestion, decontamination with N-acetyl-l-cysteine and sodium hydroxide (NALC/NaOH) and centrifugation 3500g for 15 minutes. Then, the specimen is
resuspended in 1 mL buffer phosphate sterile (pH = 6.8). The
processed specimen is then being inoculated into a solid medium (LJ) with a
volume of 0.2 mL and liquid media such as MB/BacT (bioMe´rieux, Marcy
l’Etoile, France) and Bactec MGIT 960 with a volume of 0.5 mL. Lastly, the
specimen is incubated at 37 0C for 6 weeks.6,23

The culture of M. tuberculosis is
able to detect 10-102 organism/mL specimen and has higher
sensitivity than AFB staining. However, the growth M. tuberculosis is very slow; thus, culture requires 8 weeks of
incubation.1 In
addition, it also needs a proper laboratory infrastructure and skilled staff.7,22,24

Tuberculin skin test uses a substance called tuberculin which is 200 purified
protein derivate (PPD) that can induce a delayed-type hypersensitivity after
being administered under the skin. A positive in vivo reaction showed the diameter of induration about 5 mm in the immunocompromised patient
and 10 mm in
immunocompetent children and adults who never received vaccination and 15 mm in
those who have been vaccinated. The limitation of this test is that it has poor
sensitivity, specificity, and there is a possibility of cross-reaction with
other antigen(s).11,19 Another
limitation is that it can cause false positive result in a person who has been
vaccinated with Bacilli Calmette-Guerin (BCG).11

Interferon-gamma
release assay IGRA is a method of diagnosis performed by measuring the immune
response of interferon-gamma (IFN-?) to TB antigens including early secretory antigenic
target-6 (ESAT-6), cultures filtered protein-10 (CFP-10), and TB7.7. Those
antigens were encoded by the gene in a region of difference-1 (RD1) of M. tuberculosis complex. Because
this method detects the host immune response to TB antigens located in the
specific regions of the bacterial gene, the method has higher sensitivity and specificity
than
TST.11,19

The IGRA method is based on enzyme-linked immunosorbent assay (ELISA) and
ELISpot 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. The
specificity of IGRA is higher than TST so IGRA is a very useful method for the
screening of tuberculosis, TB/HIV coinfection, and TB in immunocompromised
patients. The limitation of IGRA is due to the availability of the mononucleated
cells in peripheral blood that can affect the result and give false positive or
negative.11

The
last method for TB detection is molecular detection. The method has a rapid
turn-around time so it can overcome the slow-growing bacteria in cultivation.11 A novel and advanced technique
for TB molecular detection is GeneXpert MTB/RIF assay. The assay is a qRT-PCR
based molecular test that detects the presence of M. tuberculosis within less than 2 hours of analysis.11,25 This assay was endorsed by WHO
in 2010 as the rapid diagnosis test to detect TB and rifampicin resistance.2,26

GeneXpert MTB/RIF is an
automatic cartridge-based assay that has many benefits. First, the closed
amplification system can reduce the potency of cross-reaction. Second, the
GeneXpert assay can give information about rifampicin resistance coded in rpoB
gene. The rpoB gene is responsible for 96% of rifampicin resistance cases in M. tuberculosis infection. The
rifampicin resistance is a key predictor for multidrug-resistant
tuberculosis  (MDR-TB) as most of the
bacteria resistant to rifampicin is also resistant to isoniazid.12

GeneXpert MTB/RIF processes
the 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 that
controls the liquid inside the cartridge and performs a qRT-PCR analysis. The GenXpert instrument amplifies
the 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 not
report non-tuberculous mycobacterium (NTM) and to maximize mutation detection as the
predictor of MDR-TB.12,24

M. tuberculosis is detected using five overlapping molecular probes (A–E) in region 81-bp rpoB. It is
identified with at least two of the five probes shows positive signals with cycle threshold of 38 cycles or
less.
A cycle threshold (CT) is a
number of cycles needed to obtain the positive result. The range of CT is
classified into high, <16; medium, 16–22; low, 22–28; very low, >28.13

The sample reagent contains
sodium hydroxide (NaOH) and isopropanol with the ratio of 3:1 is added to 0,5
mL  sample.7,26 The sample reagent is used to dilute the sample, to
reduce the infectiousness of the sample, and to inactivate PCR inhibitors.6 The container consisting specimen is agitated twice
within 15 minutes and incubated at room temperature pressure (r.t.p).22,26 A volume of 2 mL mixture is inserted into the
cartridge and then the PCR assay is run by GeneXpert automatically to detect M. tuberculosis and rifampicin
resistance (RIF).6 The result is ready within less than 2 hours.26 In pulmonary TB, the assay can be done using either
raw sputum or pellet sputum.7,12

During the GeneXpert assay, there is a chance to obtain false negative or
positive results. False positive results, often caused by false amplification
target,  whereas false negative results often
related to the liquid problem or sample inhibitor.12 The error
of sample processing is reduced by using Bacillus
globigii
spore as
the internal control. The spore is inserted into the cartridge to ensure the
lysis M. tuberculosis and to detect PCR inhibitors.28 The GeneXpert instrument requires a
stable electricity, appropriate temperature (<30 0C), and routine calibration of the cartridge module.5,7 The time difference of diagnosis 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 liquid chromatography. aafter detection of growth Adopted from Diagnosis of TB in Adults and Children, IDSA Guideline.19   SAMPLES Tuberculosis can be divided into pulmonary and extrapulmonary. It is estimated that 15-20% of extrapulmonary TB affects the lymph nodes, meninges, kidney, spine, and bone. Extrapulmonary TB (EPTB)  is an atypical clinical presentation that often stimulated by neoplasia or inflammation.29 For diagnosing EPTB, the specimens can be obtained from pleural fluid, aspirate or tissues from lymph node, or CSF.30 Either pulmonary or extrapulmonary TB, there are some specimens that can be subjected to GeneXpert assay. It is important to note, however, that specimens from the outside of respiratory system contain a low number of bacilli. The specimens microbiology diagnostics should be collected from sterile locations. SR is added to ratio 2:1 except for CSF. The collected sample is then incubated at room temperature for 15 minutes.  Following incubation, the sample is inserted into the cartridge and tested using GeneXpert.29 Lymphadenitis TB is the most frequent EPTB form. The sample can be obtained from the tissue or aspirated using fine needle biopsy from the lymph node. The use of fresh sample will give higher sensitivity than the frozen sample.30 In pleural TB, the sample is taken from pleural fluid whereas meningitis TB can be diagnosed by using CSF sample. A review by Denkinger et al. (2014) shows that the use of  GeneXpert has high sensitivity to diagnose lymphadenitis TB, medium sensitivity in the diagnosis of meningitis TB and low sensitivity in pleural TB.8 Another research that used various samples to diagnose EPTB shows different sensitivities. The sensitivity of the assay when using tissue 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 values are obtained when using samples from pleural fluid (44.4%) and other body fluids such as pericardium, peritoneum and synovial fluid (50%).8 Sputum is the most frequent sample used to diagnose pulmonary TB. Sputum sample can be differentiated into raw and pellet sputum. Gastric aspirate can be used as GeneXpert sample from old patients who cannot expel sputum effectively.31 Another sample that can be used is a fecal specimen. The required volume of specimen is 2 cm3 and then 10 mL added with distilled water and then it is homogeneously using vortex.31 The mixture of the specimen and distilled water is incubated at room temperature 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% for 15 minutes at room temperature. The next step, the sample is mixed with 40 mL of phosphate buffer with pH of 6,8 and then centrifuged 3000 × g for 20 minutes. The sediment is then resuspended in 3 mL of phosphate buffer and then inoculated in Ogawa medium and MGIT. The remaining sample can be stored at ?80 °C for 1-3 months.31 Raw sputum and pellet sputum can be used to examine using GeneXpert as the recommendation from Cepheid. The pellet sputum is a raw sputum that is processed by liquefaction, decontamination using NALC/NaOH and centrifugation.6 The raw sputum can be obtained either spontaneously expectorated by the patients or after sputum induction. The required volume of pellet sputum is 0.5 mL and raw sputum is 1 mL.28 The types of sputum influence its storage and transport. Pellet sputum can be stored at 2–8 °C for up to 7 days while raw sputum can be stored at the maximum temperature of  35 °C for 3 days and then moved into 2–8 °C for the next 7 days. The sample reagent is used to liquefy raw and pellet sputum with the ratio of 2:1 and 3:1, respectively.28 Previous research reported that the sensitivity of GeneXpert using raw or pellet sputum is similar.6 CONCLUSION Tuberculosis as 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. The microscopic examination and tuberculin skin test have poor sensitivity. Interferon-gamma release assays with a better specificity than TST is affected by mononucleated cells. GeneXpert works rapidly and automatically so it can increase TB detection using various kinds of specimens regarding the site or organ. 

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