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A systemic review on tuberculosis

Article  in  The Indian journal of tuberculosis · February 2020

DOI: 10.1016/j.ijtb.2020.02.005

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indian-journal-of-tuberculosis/

Review article

A systemic review on tuberculosis

Arvind Natarajan a, P.M. Beena a, Anushka V. Devnikar b, Sagar Mali c,*

a
Department of Microbiology, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and
Research, Kolar, India
b
Department of Microbiology, S Nijalingappa Medical College, Bagalkot, India
c
SDM Narayanaya Heart Centre, Sri Dharmasthala Manjunatheshwara Medical College, Sri Dharmasthala
Manjunatheshwara University, Dharwad, India

article info abstract

Article history: Tuberculosis (TB), which is caused by bacteria of the Mycobacterium tuberculosis complex,
Received 3 December 2018 is one of the oldest diseases known to affect humans and a major cause of death world-
Received in revised form wide. Tuberculosis continues to be a huge peril disease against the human population and
7 September 2019 according to WHO, tuberculosis is a major killer of the human population after HIV/AIDS.
Accepted 18 February 2020 Tuberculosis is highly prevalent among the low socioeconomic section of the population
Available online 28 February 2020 and marginalized sections of the community. In India, National strategic plan (2017e2025)
has a national goal of elimination of tuberculosis by 2025. It requires increased awareness
Keywords: and understanding of Tuberculosis. In this review article history, taxonomy, epidemiology,
Tuberculosis pathogenesis histology, immunology, pathogenesis and clinical features of both pulmonary tuberculosis
Immunology (PTB) and extra-pulmonary tuberculosis (EPTB) has been discussed. A great length of
Tuberculosis diagnosis detailed information regarding diagnostic modalities has been explained along with
Tuberculosis treatment diagnostic algorithm for PTB and EPTB. Treatment regimen for sensitive, drug resistant and
extensive drug resistant tuberculosis has been summarized along with newer drugs rec-
ommended for multi drug resistant tuberculosis. This review article has been written after
extensive literature study in view of better understanding and to increase awareness
regarding tuberculosis, as a sincere effort that will help eliminate tuberculosis off the face
of the earth in near future.
© 2020 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

sometimes at a tardy sluggish pace, but, slow or quick, is ever

1. Introduction sure and certain …”

Charles Dickens: Nicholas Nickleby.

“A dread disease in which the struggle between soul and body is Till date the words of Charles Dickens are true. Tubercu-
so gradual, quiet and solemn and the result so sure that day by losis; A scourge of the mankind from time immemorial, the
day and grain by grain, the mortal part wastes and withers dread disease was called consumption in Dickens time had a
away. A disease … which sometimes moves in giant strides and profound social and economical effect on human existence

* Corresponding author.
E-mail address: sagar5838@gmail.com (S. Mali).
https://doi.org/10.1016/j.ijtb.2020.02.005
0019-5707/© 2020 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.
296 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1

worldwide.1 Mankind has seen changing face of tuberculosis detection using PCR sequencing in a Neolithic infant and
(TB): from an incurable disease to the curable one. With the women from 9 thousand year old settlement in the Eastern
emergence of HIV/AIDS epidemic (1981); the cursed and Mediterranean is the oldest strong evidence available. Galen
deadly co-infection of HIV and TB resulted in a global resur- (131e201) first suspected that TB could be contagious. It took
gence of TB.2 In the early 1990's, a drug resistant TB strain many centuries until Girolamo Fracastorius (1483e1553)
caused an outbreak in New York, killing 80% of infected pa- showed that some diseases could be transmitted through
tients.3 The HIV and TB co-infection, and spread of drug ‘particles’ by direct or indirect contact between humans.
resistant TB has worsened the scenario to an extent that, TB Thomas Willis (1621e1675) first described miliary TB. Calm-
has been declared a global emergency in 1993 by WHO.2 The ette extracted a protein (tuberculin) from large cultures of the
tuberculosis has varied presentation and it is divided into bacillus and first used for therapy known as ‘tuber-
Pulmonary TB (PTB) and extrapulmonary TB (EPTB) based on culinisation’, which failed as treatment for TB. The Tuberculin
clinical manifestation.4 EPTB is defined as TB involving organs was also used for intradermal skin test which was described
other than the lungs (e.g. pleura, lymph nodes, abdomen, by Charles Mantoux & used in the diagnosis of TB. Later this
genitourinary tract, skin, joints and bones, or meninges). If a intradermal skin test was named after Charles Mantoux and
patient with EPTB also has tubercular lesion in lung paren- is known as Mantoux test.15 Benjamin Marten (1690e1752)
chyma, then the patient is categorized as pulmonary TB (e.g. hypothesized that TB is caused by ‘wonderfully minute
military TB).2 If the patient suffers from intra-thoracic medi- living creatures’ in his theory of ‘contagious living fluid’. It
astinal and/or hilar lymph node TB or TB pleural effusion was Jean Antoine Villemin (1827e1892), a French army
without radiographic abnormalities in the lung is categorized doctor who successfully demonstrated the transmission of
as EPTB.5 WHO estimates shows that globally there were 10.4 TB from humans to animals and from animals to animals. In
million cases of TB, in 2017, of which two thirds were in eight 1834, Johann Lukas Schonlein proposed the name ‘Tuber-
countries: India (27%), China (9%), Indonesia (8%), the culosis’ which is derived from Latin word ‘tubercula’
Philippines (6%), Pakistan (5%), Nigeria (4%), Bangladesh (4%) meaning ‘a small lump’ seen in all forms of the disease.15 On
and South Africa (3%).6 EPTB constitutes about 15e20% of all 24th March 1882, Robert Koch announced in the meeting of
TB cases. With HIV pandemic, the EPTB scenario is further the Berlin Society of Physiology that he had discovered
complicated, as EPTB constitutes more than 50% of all cases of causative agent responsible for pulmonary TB and named it
TB in HIV positive patients.7 Due to its variety of presentation as ‘tuberkel virus’ in his paper published 2 weeks later. First
EPTB often poses a great difficulty in early diagnosis. It may innovative decision of staining tuberculosis bacilli and
present with constitutional symptoms such as fever, second innovative decision of culturing it on solidified cow
anorexia, weight loss, malaise and fatigue.8 In India, the only or sheep serum gave Robert Koch the Nobel prize of medi-
presentation may be fever of unknown origin due to its remote cine in 1905. Leon Charles Albert Calmette (1863e1933) and
infection site.9 Camille Guerin (1872e1961) developed vaccine against TB by
Among the available modalities tuberculin skin test (TST), sub-culturing Mycobacterium bovis for more than 200 times in
interferon gamma release assay (IGRA) and serological tests the Guinea pig model between 1908e1921.2,15 Arvid Wallg-
are not recommended in diagnosis of TB or initiation of Anti ren, a professor from Royal Caroline medical institute,
Tuberculosis Treatment (ATT).10 Smear examination with Sweden described clinical manifestations of tuberculous
sensitivity ranging from 10 to 37% and culture on Lowenstein infection in an article titled ‘The timetable of Tuberculosis’
Jensen (LJ) media with variable sensitivity ranging from 12 to which helped in better understanding course of TB illness.16
80% in different body fluids, also requiring 8 weeks of incu- The effective treatment for TB became a reality after the
bation for maximum sensitivity adversely affects the treat- discovery of antitubercular drugs like Streptomycin, Para-
ment plan by delaying it or subjecting patients to amino salicylic acid (PAS) and isoniazid by the mid-1940s.
inappropriate empiric therapy.11e14 This review article covers By late 1970 it was believed that TB may no longer be a
history, taxonomy, epidemiology, immunology, pathogenesis, public health problem in the developed world. But the
clinical features of pulmonary TB and EPTB, all available emergence of Acquired Immune Deficiency Syndrome
diagnostic modalities followed by diagnostic algorithm and (AIDS) in the early 1980s has ended this optimism and led to
treatment to help understand tuberculosis. the resurgence of TB worldwide.2

2. History 3. Taxonomy and description of the genus

TB or illnesses resembling TB have been described from Mycobacterium tuberculosis belongs to

different civilization since ancient times. The earliest such
description can be found in Vedas, where TB was referred to as ORDER- Actinomycetales
Yakshma meaning wasting disease. Greek, Chinese and CLASS- Actinomycetes
Arabic literature also describes TB like disease.2 Mycobacte- FAMILY- Mycobacteriaceae
rium exists on earth since last 150 million years. Typical GENUS- Mycobacterium
tubercular vertebral lesions were seen in mummies from the
Egyptian pre-dynastic era and Peruvian pre-Colombian era. Genera closely related to Mycobacterium are Gordomia,
The first weak evidence of TB in humans is from a bone lesion Tsukamurella, Nocardia and Rhodococcus.
found in a 500 thousand year old skull in Turkey. Human TB Features of genus Mycobacterium are summarized in Table 1.
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1 297

resistant tuberculosis (XDR-TB) posing a threat to TB control

Table 1 e Salient features of Mycobacterium genus.17,18
program globally.
Features
Mycobacteria aerobic, non-spore forming, non-motile
Shape slightly curved or straight rods 5. Pathogenesis
Size 0.2e0.6 mm by 1e10 mm
Colony morphology varies from species to species, ranging
The majority of droplet nuclei containing MTB from infec-
from rough to smooth and from non-
tious patients are trapped in upper airway and expelled by
pigmented to pigmented (carotenoid
pigment) ciliated mucosal cells: only a fraction reaches alveoli. The
Cell wall N-acetyl muramic acid mycobacteria then bind to cell surface of alveolar macro-
High content of Mycolic acid (70e90 phages through complement receptors, mannose receptor or
carbon atoms)-renders acid fastness type A scavenger receptor. Following phagocytosis, myco-
DNA High G þ C content (61e71 mol %) bacteria reduce acidity in phagosome and a cell wall
Generation time Slow- ranging from 20 hours to 36 hours
component (i.e. lipoarabinomannan) impairs Caþ/calmod-
for Mycobacterium Tuberculosis
ulin pathway thus inhibiting phagosome-lysosome fusion.
Following successful arrest of phagosome maturation, the
multiplication of bacilli begins and the macrophage even-
tually ruptures to release its bacilli, which are taken up by
4. Epidemiology macrophages and continues infection cycle further
expanding the spread.22 During primary infection, MTB
M. tuberculosis bacilli have infected nearly 1/3rd of the world's bacilli undergo hematogenous and lymphatic dissemination
population with 10% lifetime risk of developing TB disease.19 involving hilar and mediastinal lymph nodes forming pri-
Globally 10.4 million cases of TB reported in 2017, account- mary Ghon's complex. Eventually bacilli enter blood stream
ing to 133 cases/1,00,000 population, of which 90% of cases and reach various organs. This lympho-hematogenous
were adults (aged
15 years), 64% were male, 9% were people dissemination results in extrapulmonary tuberculosis dur-
living with HIV (72% of them in Africa) [Fig. 1]. An estimated ing primary infection or later in life during reactivation of
558 000 new cases (range- 483 000e639 000) of Rifampicin disease.23 EPTB can involve any site in the body & the most
resistant TB (RR-TB), of which almost half were in three common site is lymph node. However pleural, neurological,
countries: India (24%), China (13%) and the Russian Federation synovial, pericardial, abdominal, genitourinary involvement
(10%).6 Among 0.8 million new EPTB cases reported worldwide has been described.
(2013), maximum cases were from India accounting for 0.35
million cases.20 In India, according to Revised National a. Tubercular Lymphadenitis
Tuberculosis Control Programme (RNTCP) data, the preva-
lence of EPTB is 50% in HIV infected patients and 15e20% in From ancient times, lymph node TB has been called
non-HIV patients. The distribution of EPTB was in lymph node Scrofula or King's evil. It constitutes nearly 35% of EPTB cases.
47%, pleural cavity 30%, abdomen 10%, bones and joints 8%, Cervical lymphadenitis is the most common and reported in
CNS 2% and others 3%.21 Between 2000 and 2017, TB mortality 60e90% of tuberculous lymphadenitis cases. Involvement of
rate reduction is 42%. TB incidence has fallen by an average of cervical lymph node is due to spread of bacilli from primary
2% per year and case fatality rate of 16% in 2017, down from focus of infection in Ghon's complex or from tonsils, adenoids,
23% in 2000.6 In 30 high burden countries, India has managed sinonasal/osteomyelitis of the ethmoid bone. Initially, MTB
to reduce the prevalence rate by 50% as set by Stop TB Part- bacilli multiply in lymph node causing marked hyperemia,
nership Programme. Drug resistant TB has been reported from swelling, necrosis and caseation of involved lymph node. The
early days of introduction of ART, but multidrug-resistant inflammation, progressive swelling and matting of other
tuberculosis (MDR-TB) and more recently extensively drug nodes around, resulting in adhesion to adjacent skin and

Fig. 1 e Shows an estimated number of incident TB cases and TB deaths (in millions) from 2000 to 2017.6
298 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1

rupture into surrounding tissue or through skin forming si- esophagus, sigmoid colon and rectum. Hepatobiliary, splenic
nuses. Mediastinal lymphadenitis can compress major blood and pancreatic TB are rare and associated with miliary
vessels, phrenic nerve or recurrent laryngeal nerve or cause tuberculosis; often diagnosed in immunocompromised pa-
erosion of bronchus which is commonly seen in children. tients. MTB bacilli gain entry to abdominal organs by two
Peripheral tuberculosis of lymph nodes is classified by routes and cause disease due to reactivation of dormant focus.
Jones and Campbell into- As a result of hematogenous spread from primary lung
infection in children and as a part of miliary TB. Through
STAGE I:-Enlarged, firm, motile discrete nodes. ingestion of contaminated food and milk which infect Peyer's
STAGE II:-Large rubbery nodes fixed to surrounding tissue. patches and are transported to mesenteric lymph node, where
STAGE III:-Central softening due to abscess formation. they remain dormant.27,28
STAGE IV:-Collar stud abscess formation.
STAGE V:-Sinus tract formation.24 d. Central Nervous System (CNS) TB

b. Pleural TB It is serious and often fatal form of EPTB, predominantly

affecting young children. CNS TB is difficult to diagnose. It
The incidence of pleural TB is as high as 30% of all EPTB presents in 2 major forms
cases in high burden countries. The patients usually presents
with acute febrile illness with nonproductive cough and pleu- i. TB meningitis- 0.5e1% of all TB cases
ritic chest pain; associated with night sweats, chills, weakness, ii. Intra-cranial tuberculoma-accounting up to 40% of brain
dyspnea, weight loss. The pathogenesis in pleural TB is pre- tumors
sumed to be due to delayed hypersensitivity rather than direct
infection of pleural space. This space is infected from initial MTB bacilli reach CNS during dissemination that occurs in
lung parenchymal lesions and results in immunological active pulmonary disease. These bacilli cross physiological
response predominated by neutrophils (first 24 hours). This is Blood Brain Barrier (BBB) via infected monocytes/neutrophils
followed by lymphocyte driven immune response forming and cause a caseating focus in brain parenchyma or
pleural granuloma formation and release of Adenosine Deam- meninges. These foci are termed as ‘Rich foci’. Later, these foci
inase (ADA). Neutrophils remain the first line of defense for first rupture in subarachnoid space triggering inflammatory T cell
24 hours, followed by macrophages which peak at 96 hours and response with elevated levels of INF g and TNF-a in CSF. The
then by lymphocytes. A strong T-helper type-1 (Th 1) response subsequent inflammation leads to production of inflamma-
is necessary to contain MTB. Activated CD3þ and CD4þ Th1 tory infiltrates which obstructs CSF outflow causing hydro-
cells release interferon g (IFN- g) thus activating macrophages cephalus and vasculitis leads to infarction, causing potentially
to kill MTB. The Th1 immunity in pleural TB is confirmed by the irreparable neurological damage.29
high levels of IFN- g, interleukin-12 (IL-12) and elevated helper T
cells in pleural fluid as compared to serum/peripheral blood. e. Bone and Joint TB
The delayed hypersensitivity reaction to mycobacterial anti-
gens affects pleura and increases the permeability of pleural It accounts for 10e15% of all EPTB cases. It arises from
capillaries, and thereby increasing fluid in pleural cavity. The reactivation of dormant MTB bacilli lodged in any bone (spine
fluid is drained through openings in the parietal pleura called or large joints) during bacteremia of primary lung infection.
stomata. Since diffuse involvement of parietal pleura with TB These bacilli have affinity for spine and large joints because of
and damage to or obstruction of stomata leads to accumulation their rich vascular supply. An extension of initial infection
of pleural fluid. Chronic TB empyema resolve leaving thick- focus from the bone to the joint results in tuberculous
ened, scarred and calcified pleura causing chronic chest pain, arthritis. Rarely the bacilli can reach spine from the lung along
dyspnea and impaired lung function. Pleural fibrosis, a well- the Batson paravertebral venous plexus or by lymphatic
documented complication has been reported in 5e55% of drainage to the paraaortic lymph nodes. Non tuberculous
pleural TB cases.25,26 mycobacteria (NTM) have been reported to cause osteo-
articular TB following a traumatic injury or during surgical
c. Abdominal TB procedure like joint arthroplasty. NTM bone infection in pa-
tients with AIDS or transplant recipients occurs through he-
The abdominal tuberculosis is diagnosed in 11% of patients matogenous dissemination. In recent years M. bovis skeletal
with EPTB which was around 55e90% in era before effective infections have been reported in individuals who receive
ATT. The most common site of gastrointestinal tract intravesical BCG vaccine therapy.30
involvement is the ileocecal region due to following reasons-
f. Genito-urinary TB (GUTB)
i. More lymphoid tissue (Peyer's patches)
ii. Increased physiological stasis It accounts for 15% of all EPTB cases and 3e4% of all PTB
iii. Rate of fluid and electrolyte absorption is more cases. Its occurrence is 20 times more in kidney transplant
iv. Low digestive activity recipients than in general population.12 After hematogenous
spread of bacilli from active site of infection (usually lungs),
Other sites of involvement in decreasing order are bacilli gets lodged in kidney (most common site of GUTB) and
ascending colon, jejunum, appendix, duodenum, stomach, form metastatic lesions (tubercles). These foci of infection
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1 299

may heal spontaneously/due to treatment, enlarge and from host's cell mediated immunity. These tubercles are
rupture into nephrons or remain dormant for many years. microscopic to begin with and coalesce to become macro-
Usually the spread of infection is descending from kidney to scopically visible granulomas. The granulomas contain MTB
other genito-urinary organs. It develops between 2nd and 4th bacilli within macrophages, fibrin rich alveolar exudate,
decades of life; usually 5e25 years of inactivity after primary lymphocytes and multinucleated giant cells which are
lung infection.31 enclosed within fibroblastic rim. These granulomas formed
are both caseating and non-caseating granulomas33 (Fig. 2A,B
g. Miliary TB and C).

Miliary TB account for less than 2% of all tuberculosis cases

and up to 20% of all EPTB cases among immunocompetent 7. Immunology
adults, however the autopsy studies have shown military TB
ranges between 0.3% and 13.3%. TH2 response plays a central Robert Koch (1880) demonstrated delayed hypersensitivity
role in immunopathogenesis of military TB. It inhibits pro- reaction in Guinea pigs and later in humans using mycobac-
tective response such as granuloma formation and fencing of terial extracts. Seifert (1934) purified MTB extract which later
the disease activity at the site of infection. The production of become reference used as Purified Protein Derivative (PPD) in
interleukin 4 (IL-4) during TH2 response, downregulates nitric tuberculin test. In 1945 M. Chase demonstrated that immunity
oxide synthase (NOS), tall like receptor 2 and macrophage against MTB cannot be transferred to animals by immune
activation; thus sabotaging protective response of TH1 cells. serum but by transfer of CD4 T lymphocytes. It is now clear
This process favors dissemination of MTB.32 that protection against MTB is through mainly T lymphocyte
activating the macrophages. Dendritic cells (DCs) present in
proximal draining lymph nodes play a major role in priming
6. Histology naı̈ve T cells. DC takes part in surveillance around airways,
vessels and in the loose connective tissue. The mycobacterial
Any site of infection involved in PTB or EPTB has pathogno- specific lipoglycan lipoarabinomannan (LAM) binds to recep-
monic lesions known as tubercles. This is characteristic tor present on DC to gain entry. The lipoid adjuvant of LAM
granulomatous inflammatory reaction against MTB bacilli activate antigen presenting cells (APC) through toll like

Fig. 2 e (A) Caseous necrosis. (B) Granuloma surrounded by lymphocytes. (C) Multinucleated giant cells & lymphocytes in
tubercular granuloma.
300 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1

receptor-2 (TLR-2). Both DC and APC ultimately prime T lym- 4. Abdominal TB

phocytes, after which memory CD4 and CD8 cells play central
role in immune response against MTB. The activated CD4 and The clinical presentation depends on site of involvement as TB
CD8 cells have ability to kill intercellular MTB through secre- can affect any location from mouth to anus. The most common
tion of cytolytic molecules (e.g granulysin, perforin) and che- site of involvement is terminal ileum or caecum and manifest as
mokines (e.g CCL5 which attracts infected macrophages). pain abdomen, a palpable mass sometimes with weight loss,
Natural killer cell (NK cells) acts as bactericidal against MTB fever and loss of appetite. Tubercular peritonitis presents with
which are part of innate immunity. NK cells do not require classic doughy abdomen, ascites, pain abdomen and fever.
APCs for their activation and also improve function of g d T In esophageal TB, additional symptoms seen are
cells. The g d T cells are mycobactericidal and potent secretors dysphagia, odynophagia and retrosternal pain/discomfort.
of INF-g like macrophages. The activated T lymphocytes Patient also suffers from life threatening complications like
release IFN g, TNF- a, and Interleukin-2 activating other broncho-esophageal fistula/hematemesis.
resting monocytes/macrophages. g IFN up regulates produc- Gastric TB is rare because of acidic pH, few lymphoid tis-
tion of TNF, toxic oxygen species and nitric oxide in macro- sues in mucosa and rapid gastric emptying. Duodenal TB
phages. These lead to granuloma formation and effective presents with dyspepsia, duodenal obstruction and duodenal
containment of MTB inside granuloma.34 ulceration. Other reported complications are perforation,
fistulae and obstruction jaundice. The common presenting
feature in rectal TB is hematochezia followed by constitu-
8. Clinical features tional symptoms and complication. It may also present as
anal fissure, fistulae or perirectal abscess.28,35
EPTB is less common when compared to PTB, thus less
commonly encountered by clinicians and difficult to diagnose 5. CNS TB
clinically.
The most common manifestation of CNS TB are meningitis
1. Miliary TB (95%), tuberculomas (2%) and abscess (1%). Clinical features
include those related cranial nerve involvement as well as
The clinical features are usually non-specific and may headache, vomiting, decreased level of consciousness, neck
present with fever, weight loss, night sweats, anorexia stiffness and in the absence of medical care coma and
and weakness. The physical findings in descending order death.35,36
are fever, wasting, hepatomegaly, pulmonary findings,
lymphadenopathy and splenomegaly. A granuloma in 6. Skeletal TB
retinal choroid is strong suggestive feature of disseminated
TB.32,35 Pain is the most common presenting feature. The involved
joints have limited motion of range with or without the pres-
2. TB Lymphadenitis ence of swelling. The patient may present with sinus tract.
Involvement of spine leads to chronic backache, fever and
It presents as painless swelling in cervical region (supra- more than 50% of patient suffer from neurological symptom
clavicular fossa). Usually the process is bilateral and with due to compression of spinal cord. Delayed diagnosis may
progression of disease, the lymph node fuse and become further complicate the situation due to spinal deformity and
matted. The overlying skin gets inflamed, ultimately enlarged severe, irreversible neurological sequelae like paraplegia.30,35
lymph node rupture through inflamed skin forming sinus
tract. Intra thoracic adenopathy may cause atelectasis by 7. Genito-urinary TB
compressing bronchi or bronchiectasis (common in
children).24,36 Patient usually presents with local symptoms like dysuria,
hematuria, flank pain and increased frequency of micturition.
3. Pleural TB In women, genital involvement presents with pelvic pain,
menstrual irregularities and infertility whereas in men the
The presentation in tubercular pleurisy depends on num- most common presentation is scrotal swelling/mass with or
ber of bacteria infecting pleural space. If few MTB bacilli gain without pain. Symptoms of prostatitis, orchitis or epididy-
entry into pleural space, then it leads to hypersensitivity mitis may also occur depending on site of involvement.31,35
response causing pleural effusion. The process may resolve
spontaneously or may lead to large effusion causing fever,
pleuritic pain, dyspnea and weight loss. If large number of 9. Lab diagnosis
MTB bacilli gain entry from rupture of a cavity or the adjacent
parenchymal fistula, then it leads to tubercular empyema. The Definitive diagnosis of tuberculosis involves demonstration of
presentation of pleural TB in HIV seropositive patients is M. tuberculosis bacilli by microbiological, cytopathological or
chronic with additional symptoms like tachypnea, night histopathological methods.2 The classic laboratory approach
sweats, fatigue, diarrhea and have more hepatomegaly, to the diagnosis of mycobacterial infections involves the
splenomegaly, lymphadenopathy as compared to seronega- phenotypic characterization of colonies growing on
tive patients.26,35 LowensteineJensen medium. The current recommendation is
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1 301

that a combination of phenotypic and molecular assay are Acid alcohol-add 3ml of concentrated HCL slowly to 97ml
used for the rapid identification of mycobacteria, particularly of 90e95% ethanol.
for the identification of M. tuberculosis.37 Accurate diagnosis of Methylene blue counterstain-dissolve 0.3g of methylene
EPTB depends on the detection of mycobacteria by using blue chloride in 100ml of distilled water.
direct and indirect approaches.7 The tests used in the diag- Examine with 100 oil-immersion objective. Mycobacteria
nosis of tuberculosis are listed in Table 2. are stained red and the background light blue.
The heating process during staining helps to penetrate
9.1. Microscopy carbol fuchsin into the MTB bacilli. The smear is decolorized
using 20% H2SO4 and counter stained with methylene blue.
Mycobacteria are recovered from variety of pulmonary and MTB bacilli resist decolourisations by 20% H2SO4 hence are
extrapulmonary samples. At least 10 000 AFB should be known as AFB.37
present per ml of sample for them to be readily demon- Ziehl-Neelsen staining is reliable, reproducible and cost
strable in direct smears.38 Microscopy is reliable, repro- effective and also useful in monitoring response to anti TB
ducible, inexpensive, an indicator of infectiousness, a treatment. Smear microscopy has low and variable sensitivity
comprehensive tool for diagnosis/monitoring progress/ values (0e40%) and could not differentiate between Mycobac-
defining cure and even feasible in the remote or tribal terium tuberculosis and nontuberculous mycobacteria.40e42
places.39
WHO evaluation showed that the diagnostic accuracy of 2. Kinyoun (cold stain):-
light e emitting diode (LED) microscopy is comparable to that
of conventional fluorescence microscopy with much less The reagents and dyes used are-
expense.7 RNTCP has provided light emitting diode based Carbol fuchsin-dissolve 4g of basic fuchsin in 20ml of
fluorescent microscope services in 200 medical colleges across 90e95% ethanol and then add 100ml of 9% aqueous solution of
India as a pilot study.39 phenol (9g of phenol dissolved in 100ml of distilled water)
Acid alcohol-add 3ml of concentrated HCL slowly to 97ml
9.1.1. Acid fast staining of 90e95% ethanol.
The cell wall of mycobacteria, because of their high lipid con- Methylene blue counterstain-dissolve 0.3g methylene blue
tent, have the unique capability of binding the Fuchsin dye so chloride in 100ml of distilled water.
that it is not removed (destained) by acid alcohol. The presence Examine with 100 oil immersion objective.
of acid fast bacilli (AFB) in the smear, combined with history of Mycobacteria are stained red and the background light
weight loss, fever, night sweats and radiological evidence of old blue. This technique is cold stain technique because increased
pulmonary lesion helps in early diagnosis. Acid fast smears are phenol concentration replaces heating step and helps in
also useful in monitoring response to treatment. penetration of carbol fuchsin.37
Types of acid-fast stains used are:
3. Auramine Fluorochrome:-
i. Carbol fuchsin stains: a mixture of fuchsin with phenol
(carbolic acid)Ziehl-Neelsen (hot stain)Kinyoun (cold stain) The reagents and dyes used are-
ii. Fluorochrome stain: Auramine O, with or without a second Phenolic Auramine-dissolve 0.1g of Auramine O in 10ml of
fluorochrome, rhodamine.37 90e95% ethanol and then add to a solution of 3g of phenol in
1. Ziehl-Neelsen (hot stain):- 87ml of distilled water. Store the stain in a brown bottle.
Acid-alcohol- add 0.5ml of concentrated HCL to 100ml of
The reagents and dyes used are- 79% alcohol.
Carbol fuchsin-dissolve 3g of fuchsin in 10ml of 90e95% Potassium permanganate-dissolve 0.5g potassium per-
ethanol. Add 90ml of 5% aqueous solution of phenol. manganate in 100ml of distilled water.

Table 2 e List of tests used in laboratory diagnosis.

Microscopy Culture Conventional methods Molecular methods
Acid fast staining Solid Media i. Rates of Growth 1. Nucleic acid probes
i. Ziehl-Neelsen a. Egg-based Media ii. Pigment production 2. In situ hybridization
ii. Kinyoun b. Agar-based Media iii. Niacin accumulation test 3. Nucleic acid amplification
c. Selective Media iv. Nitrate reduction test methods
Fluorochrome v. Tween 80 Hydrolysis 4. Line probe assay
Liquid Media vi. Catalase test 5. Transcription mediated
staining
a. MGIT vii. Arylsulfatase activity amplification
b. BACTEC 460TB system viii. Urease activity 6. DNA sequencing
c. Automated Continuous Monitoring Systems ix. Pyrazinamidase 7. Spoligotyping
x. Iron uptake 8. DNA microarray analysis
xi. Growth on MacConckey 9. CBNAAT
HPLC(for culture confirmation and speci-
xii. Growth in 5% sodium 10. PBMC circ RNA detection
ation) chloride
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Examine with 25 objective for scanning. The 40 objec- 9.2.2. Liquid media
tive is used to confirm any suspicious forms. Mycobacteria are Broth media can be used for early isolation of mycobacteria
stained yellow-orange against a dark background in fluores- and also for subsequent subculturing. Middlebrook 7H9,
cent microscope. BACTEC 12B and Dubos Tween albumin broth are commonly
The fluorochrome stain offers the advantage of greater used liquid media. 7H9 broth is used as a basal media and
sensitivity compared with the ZN stain, since a significantly Tween 80 acts as a surfactant which disperses clumps of
larger area of the smear can be scanned thus reducing the mycobacteria resulting in homogenous growth. The liquid
time needed. Fluorescent microscopy increases sensitivity by media are currently used in semi-automated and automated
10% over ZN staining.37 like BACTEC 460TB system, BACTEC MGIT 960 system, BAC-
RNTCP has supplied light emitting diode based fluorescent TEC 9000MB, ESP culture system 2 and MB/BacT ALERT 3D
microscope (LEDFM) in 200 medical colleges across India to system, BACTEC MYCO/F lytic blood culture bottle.
reduce the burden on laboratory technicians in high work load
settings (>25 slides per day).39 1. Mycobacteria Growth Indicator Tube (MGIT):-

9.2. Culture The MGIT contains a modified Middlebrook 7H9 broth in

conjunction with a fluorescence-quenching-based oxygen
Isolation of M. tuberculosis from clinical samples by culture sensor (silicon rubber impregnated with a ruthenium penta-
is the ‘gold standard’ for a definitive diagnosis of TB. Culture hydrate) to detect growth of mycobacteria. The presence of
methods are much more sensitive because fewer bacilli oxygen in the medium quenches the fluorescence of the
(10e100 bacilli/ml of concentrated material) can be detected sensor. As mycobacteria or other organisms grow in the broth,
and provides the necessary isolates for conventional drug leads to depletion in the oxygen level and the indicator fluo-
susceptibility test and species identification. The sensitivity resces brightly when illuminated with UV light at 365nm. The
of culture for identification of M TB ranges between 0 and broth is enriched with 0.5ml of OADC (Oleic Acid, Bovine Al-
80% in different extrapulmonary specimens.9,43e45 The most bumin, Dextrose and Catalyse) and 0.1ml of PANTA antibiotic
commonly used solid media for culture of M TB is LJ media mixture (Polymixine B, Amphotericin B, Nalidixic Acid,
which usually takes 4e8 weeks for visible growth.46 Trimethoprim and Azlocillin). In BACTEC MGIT 960 system the
tubes are continuously monitored by the instrument. Sensi-
9.2.1. Solid media tivity and time to growth detection of the MGIT system are
similar to those of the BACTEC 460TB system and have been
1) Egg-based Media superior to those obtained with solid media in clinical evalu-
ation. But contamination rates are slightly higher for MGIT
It contains whole eggs or egg yolk, potato flour, salts and system than for BACTEC 460TB system.18,47
glycerol are solidified by inspissation. They have good buffer
capacity and a long shelf life and support good growth of most 2. BACTEC 460TB system:-
of mycobacteria. Of the egg-based media, L-J medium is most
commonly used in clinical laboratories. It is a semi-automated system which uses 14C-labeled
palmitic acid as carbon source in the medium and when
2) Agar-based Media metabolized by microorganisms to 14CO2, it is monitored by
the instrument. The amount of 14CO2 and the rate at which
In contrast to egg-containing media, agar based media are the gas is produced are directly proportional to growth rate of
chemically better defined. Colonies may be observed in 10e12 the organism in the medium. The average detection time for
days, in contrast to 18e24 days with egg-based media. Thinly smear positive specimen is 9e14 days in case of M. tuberculosis
poured 7H11 agar plates can grow micro colonies in 11 days and less than 7 days for NTM. The positive vials can also be
and can be examined by focusing agar surface through the used for drug susceptibility testing. The disadvantages of this
bottom of the plate at 10  to 100 magnification. This method system include inability to observe colony morphology, diffi-
is used as an alternative to broth cultures. It can also be used culty in identifying mixed cultures, overgrowth by contami-
for susceptibility testing. nants, cost and radioisotope disposal.18

3) Selective Media 3. Automated Continuous Monitoring Systems:-

The addition of antimicrobial agents may be helpful in The BACTEC 9000 MB system uses fluorescence-
eliminating growth of contaminating organisms. If a selective quenching-based oxygen sensor same as the MGIT system to
medium is used for a particular specimen, it should not detect growth. In ESP Culture System 2, growth is detected by
be used alone but in conjunction with a non-selective agar monitoring pressure changes in the headspace above the
or egg-based medium. LJ media is made selective by broth medium in a sealed bottle resulting from gas production
adding penicillin and nalidixic acid, this is called Gruft by microorganisms. The MB/BacT ALERT 3D system employs a
modification or it can be made selective with cycloheximide, colorimetric CO2 sensor in each bottle and reflected light to
lincomycin, and nalidixic acid. Mitchison selective 7H11 me- monitor the presence and production of CO2 dissolved in the
dium contains carbenicillin, polymyxin B, trimethoprim and culture media. As the microorganism grow, CO2 is generated
amphotericin B.18 which diffuses through membrane to sensor and dissolves in
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water present in the sensor causing accumulation of 9.3. Identification using conventional methods37
hydrogen ions.
i. Rates of Growth
CO2 þ H2O 4 H2CO3 4 Hþ þ HCO3
A rate of growth, the time of recovery varies from media to
The amount of CO2 produced is proportional to the growth media-the average time of recovery of mycobacteria on egg
of microorganism in the media, as the CO2 levels increase, the based media is about 21 days, but ranges from as short as 3e5
concentration of hydrogen ions increases, thus reducing the days to as long as 60 days depending on the species. The growth
pH of sensor causing color change from dark green to light of micro colonies on 7H10 agar is detectable from 3 to 12 days.
green or yellow. In a study the average time to detection of micro colonies of
A light emitting diode system projects light on the sensor MTB was 11 days on middlebrook 7H11 agar, 16 days with MB/
and the reflected light is measured by photodetector. The BacT bottles and 19.5 days with LJ media. Some mycobacterial
color change of sensor increases reflectance units which is species belonging to rapid grower group grows within 7 days on
monitored and recorded by the instrument to determine the LJ media.
positive or negative result.18
BacT ALERT 3D system has been evaluated in many studies ii. Pigment production
for rapid detection of growth and also for DST. A study by
Carricajo A et al. reported mean detection time of M TB Mycobacterium species have capability of producing col-
complex from pulmonary and different extrapulmonary ony pigmentation in the dark (scotochromogen) or only after
samples were 22.8 days with LJ medium and 16.2 days with the exposure to light (photochromogen) doesn't finalize species
BacT ALERT 3D system.47 In study by Piersimoni et al. Mean identification but narrows possibilities. Even after exposure to
detection time of M TB complex in smear positive samples by light MTB fails produce pigment, beyond a light buff color.
BacT ALERT 3D, B460 and LJ media was 11.5, 8.3 and 20.6 days Non tuberculous mycobacteria
respectively whereas in smear negative samples it was 19.9, Non tuberculous mycobacteria (NTM) has been classified on
16.8 and 32.1 days respectively.49 the basis of pigment production and rate of growth by Runyon-
These studies have reported that mean detection time
taken by BacT ALERT 3D system was 16e18 days compared to GROUP I- Photochromogens
22e32 days by using LJ media for M TB complex. Thus BacT GROUP II- Scotochromogens
ALERT 3D system reduces detection time by 25%.48e50 In GROUP III- Nonchromogens
another study by Moore WAJ et al. the median time to culture GROUP IV- Rapid growers
positive was 7 days, 13 days and 26 days for microscopic iii. Niacin accumulation test
observation drug susceptibility culture, automated culture
and LJ culture respectively.51 All mycobacteria produce niacin, but only MTB and M.
The BACTEC MYCO/F LYTIC culture bottle has lytic agent to simiae lack the enzyme required to further convert the niacin
release mycobacteria phagocytosed by white blood cells. The to niacin ribonucleotide. Reagent impregnated filter paper
incubation and monitoring is similar to other BACTEC blood strips incubated in test medium produces yellow color which
culture bottles. It can also be used to culture other bacteria is indicative of niacin accumulation.
and fungi present in blood stream.37
iv. Nitrate reduction test
9.2.3. Gas-liquid and high-performance liquid
chromatography (HPLC) MTB produces nitroreductase which catalyzes the reduc-
Analysis of fatty acids by gaseliquid chromatography is a tion of nitrate to nitrite. Production of red color after adding
rapid and reliable method for the culture confirmation sulfanilic acid and N-naphthyl ethylenediamine to an extract
mycobacteria and also identification of the species. The of the unknown culture is indicative of the presence of nitrite
method is based on development of profiles of mycolic acids, and a positive test.
which vary from one species to another. This technology to
speciate mycobacteria is available only in few reference v. Tween 80 Hydrolysis
laboratories.37
Denaturing HPLC is an alternative for other methods which This test is useful in identifying mycobacteria which
utilizes a molecular probes like DNA sequence analysis (which possess lipase that splits Tween 80 into oleic acid and sorbitol.
is regarded as the gold standard for mutation detection), M. kansasii and M. gordonae hydrolyze Tween 80.
reverse line hybridization, single-strand conformation poly-
morphism, DNA macroarrays and real-time PCR. This tech- vi. Catalase test
nique is relatively inexpensive, same-day results can be
obtained, potentially any mutation in the amplified fragment The enzyme catalase splits H2O2 to release O2 which is
can be detected and it can be applied on a universal basis. indicated by the presence of effervescence. The catalase ac-
Other studies have shown this method can be useful for tivity after heating the culture at 68  C for 20 min (heat stable
detecting mutations on rpoB (RMP), katG (INH), pncA (pyr- catalase) is not seen in most strains of MTB. Semi-quantitative
azinamide), rspL (streptomycin) and embB (ethambutol) assessment of catalase activity is by measuring the height
gene.52 achieved by the column of bubbles produced when H2O2 is
304 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1

added to tube culture. A column higher than 45mm is 1. Nucleic acid probes
considered a positive test.
This is a first nucleic acid based technology used to identify
vii. Arylsulfatase activity mycobacteria in positive cultures with very high accuracy,
sensitivity and specificity. In this technique ribosomal RNA
The enzyme arylsulfatase in mycobacteria breaks tripo- (rRNA) present in the cells and in culture in high quantities acts
tassium phenolphthalein sulphate to release phenolphtha- as genetic target. The radiolabeled (acridine ester) single
lein. This free phenolphthalein turns the media pink in the stranded DNA probes hybridizes with rRNA forming stable DNA-
presence of sodium bicarbonate. This test differentiates RNA complex. After the inactivation of unhybridized probe, light
rapidly growing mycobacteria (positive) from group III non- generated is recorded by an instrument which is proportional to
photochromogenic mycobacteria (negative). the amount of probe present. A predetermined threshold is used
to determine positivity. This technique requires two hours.
viii. Urease activity
2. In situ hybridization
The presence of urease activity is used to differentiate M.
scrofulaceum (positive) from M. gordonae (negative). This technology uses an oligonucleotide probe labeled with
fluorescein and the interpretation is made by direct observa-
ix. Pyrazinamidase tion using fluorescence microscopy. It is popularly known as
fluorescence in situ hybridization (FISH). If the detection of
The enzyme pyrazinamidase deaminates pyrazinamide hybridized group is done by secondary reaction and color, the
to form pyrazinoic acid which produce a red band in the reaction is known as chromogenic in situ hybridization (CISH).
culture media. This test is useful in distinguishing MTB, M. FISH has been used to detect MTB in cultures and in direct
bovis, M. kansasii and M. marinum from other species of respiratory samples that contain AFB.
mycobacteria.
3. Nucleic acid amplification (NAA) methods
x. Iron uptake
In late 1990's FDA approved amplicor M. tuberculosis PCR
M. fortuitum and M. smegmatis take up soluble iron salts assay (Roche diagnostics) and amplified M. tuberculosis direct
from culture media to produce rusty brown appearance on test (AMTD) for respiratory specimens. These assays perform
addition of 20% ferric ammonium citrate. Other mycobacteria well on smear positive specimens but sub optimally on smear
species lack this property. negative respiratory specimens, when compared with culture.
Lately many in-house PCR and more recently real-time PCR
xi. Growth on MacConckey assays have been developed and tested.
A study by Laraque F et al. tested performance of NAA on
MacConckey supports the growth of rapidly growing respiratory samples (N ¼ 4642) and found that NAA had a
mycobacteria. However, most other mycobacterium species sensitivity of 96% and specificity of 95.3% in specimen tested
cannot grow on this media. positive for AFB on smear.53 In another study by Guerra RL
et al. the effect of NAA results in clinical care of Pulmonary TB
xii. Growth in 5% sodium chloride was evaluated. A total of 638 cases were included of which 270
were positive for MTB on culture. NAA had a sensitivity of
M. triviale and some strains of M. falvescens, M. fortuitum 92.3% and specificity of 99.8%. NAA had decreased length of
and M. abscessus can grow on egg based culture media con- unnecessary therapy from 31 days to 6 days.54
taining 5% NaCl when incubated at 28  C. Polymerase Chain Reaction:
PCR technique is now widely used in the research and diag-
9.4. Molecular methods nostic fields. This technique is based on amplification of specific
DNA sequence to a large number of copies that can be detected
There is a movement in clinical laboratories away from the by separation on gel electrophoresis. The amplification is ach-
conventional time consuming and tedious test for species ieved by using synthetic oligonucleotide primers complementary
identification of Mycobacteria recovered in culture e.g. nucleic to specific DNA sequence. This process leads to a million fold
acid probes have been produced to identify MTB, Mycobacte- amplification of target DNA through multiple cycles of:
rium avium intracellulare, M. kansasii and M. gordonae. There
are 4 major applications used in clinical laboratories: i. Denaturation
ii. Annealing
1. Use of DNA probes for culture confirmation of isolates iii. Extension
recovered from clinical specimens.
2. Use of DNA sequencing for identification of mycobacteria. This results in an exponential increase in the number of
3. Use of nucleic acid amplification tests (NAAT) for direct copies of the target. A number of target genes of mycobacterial
detection of MTB from clinical specimens. DNA have been evaluated for diagnosis by PCR and various other
4. DNA finger printing and strain typing of mycobacterium genotypic methods. The different DNA amplification targets used
species. are e IS6110, devR, rpoB, IS986 and genes encoding MPB-64
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1 305

(mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 The amplicons are applied to the strip. Line or dots are formed
(esat6), and CFP-10 (cfp 10) proteins. Any stretch of nucleic acid at the site of amplicon-probe hybridization. The pattern
can be amplified by using DNA polymerase, provided that the formed at the end of reaction is then compared with standard
specific sequence data are known to allow the designing of key to interpret the results of that particular reaction. This
appropriate primers. The target most frequently amplified is the technology is reverse of Southern blot technique with
IS 6110 repetitive element which is present in multiple copies (up advantage of numerous probes can be tested simultaneously.
to 20) in most strains of M. tuberculosis. Species specific and genus Radioisotopes are not used which is another advantage of this
specific PCR methods are being used with various targets and technology over Southern blot. These assays are relatively
modifications of PCR.55 Different studies by Abe et al.,56 Clarridge simple when compare to DNA sequencing with simpler post-
et al.,57 Beige et al.,58 Nol te et al.59 and Cheng et al.60 have shown analytic analysis. It is used for species identification as well as
sensitivity of PCR to be 84%,86%,98%,91% and 72% respectively. detection of mutations that lead to drug resistance.37

Advantages of PCR: 5. Transcription mediated amplification (TMA):

1. Most sensitive and rapid method of detection, can even detect TMA amplifies rRNA via DNA intermediate, producing billions
when the bacilli number is as less than 10 {1e10 AFB/ml} of copies of RNA within an hour followed by detection using
2. Determine rapidly whether AFB identified by microscopic acridinium ester labeled DNA probes. Results are read in a
examination in clinical specimens are M. tuberculosis or luminometer in terms of Relative Light Units (RLU). Samples with
atypical mycobacteria. values of 30 000 RLU are considered positive. TMA is sensitive
3. Identify the presence of genetic modifications known to be enough to detect as little as 2.5 femptogram(fg) of RNA in clinical
associated with drug resistance. samples. Since there are 3e5fg of rRNA per M. tuberculosis cell,
this assay is capable of detecting the rRNA contained in a single
Disadvantages of PCR: cell and is useful in conditions where culture is not feasible due
to considerable time requirement or in paucibacillary.61
1. False positive reactions-due to carry over contamination
2. False negative reactions-due to presence of inhibitors that 6. DNA sequencing:
interfere with the PCR
3. High cost This technology is very useful in the identification of slow
4. Amplification of DNA from both live and dead bacilli. So it growing organisms like mycobacteria. It is a complicated
cannot be used for monitoring therapy response. technique than simple probe hybridization and requires
5. Inhibition of amplification and reproducibility of the assay experience in sequence alignment, editing software and ge-
netic data basis. In this technique, hypervariable A region of
The effectiveness of PCR for tuberculosis depends on the 16S gene complex is most commonly targeted for rapid
experience and accuracy of the personnel conducting the and accurate identification of mycobacteria. MicroSeq
assay. In many studies sensitivity of PCR has been compared (Applied Biosystems, Inc, foster city, CA) has made this tech-
with microscopy and culture results. nology commercially available. The rpoB gene was popularly
The disadvantages of PCR were addressed in a multi- used as sequencing target because it provides identification
laboratory study conducted by Noordhoek and coauthors. information as well as information about the susceptibility to
They had sent 200 sputum, saliva and water samples con- Rifampin.37
taining known numbers of M bovis BCG along with negative
controls to 7 laboratories. Each laboratory used IS6110 inser- 7. Spoligotyping:
tion sequence as the target and their own protocol for PCR.
High levels of false positive results ranging from 3% to 20% This technique is used for studying genetic diversity and
were reported. This was due to lack of monitoring of each step epidemiologic study of M TB strain circulating in a particular re-
of the procedure. To overcome these problems there is a ne- gion. In this technology, DNA polymorphism is detected at direct
cessity of careful quality control during every step of assay. repeat locus (DR locus) of M TB genome by hybridization assay
Real time PCR is a technique that reduces the detection which is then used for phylogenetic analysis. The predominant
time and also quantifies the amount of M TB present in the spoligopatterns reported from Indian studies are CAS, EAI, Beijing,
clinical sample. The whole process of amplification and manu. The technique further characterizes MTB strain and its
detection takes place in single reaction vessel in a closed importance in determining clinical manifestation.62
system. Thus it reduces risk of amplicon contamination of
laboratory. Since this technique is completely automated 8. DNA microarray analysis:
there is no need of post amplification processing and elec-
trophoresis for detection of amplicons.37 High density oligonucleotide arrays (DNA microarrays)
offer the possibility of rapid examination of large amounts of
4. Line probe assay DNA sequences with a single hybridization step. This
approach has recently been applied to simultaneous species
It is a reverse-hybridization technology made available identification and detection of mutations that confer drug
commercially by Innogenetics and Roche diagnostics. It uses resistance in mycobacterium. The DNA microarray is very
nitrocellulose strip on which multiple probes are immobilized. promising method for the future because of following
306 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1

i. Easy to perform 10. PBMC circRNA detection

ii. Can be readily automated and
iii. It allows for identification of a large number of myco- In a study by Quin Z et al., peripheral blood mononuclear
bacterial species in one reaction. This technique is cell (PBMC) circular RNA (upregulated disproportionately) was
expensive and currently used only for research work.37 used as diagnostic liquid biomarker to diagnose active pul-
monary TB. In the study, it was found that PBMC circRNA
9. CBNAAT(Cartridge Based Nucleic Acid Amplification
levels were significantly higher within 5 pathways namely
Test)
“cytokineecytokine receptor interaction”, “chemokine
signaling pathway”, “Fc gamma R-mediated phogocytosis”,
GENEXpert MTB/RIF assay:
“neurotrophin signaling pathway” and “bacterial invasion of
WHO has recommended GENEXpert MTB/RIF assay for the
epithelial cells”. The levels were increased in both young and
early diagnosis of TB and for the detection of resistant to
old TB patients. A circRNA signature was developed based on
Rifampicin in 2011. This simple cartridge based nucleic acid
above mention dysregulated pathways and was further vali-
amplification test has revolutionized TB control program. It
dated by qRT-PCR, confirmed by microarray in 10 TB patients
requires only 130 TB bacilli per ml of sputum for a positive
and 11 healthy controls. This technique has potential to be
result. It is also used for pleural, peritoneal, cerebrospinal,
used as new tool in the diagnosis of active pulmonary TB.67
pericardial, and synovial fluid samples thus improving the
Extensive and detailed information regarding available as
diagnosis of extrapulmonary tuberculosis and detection of drug
well as outdated diagnostic tests has been explained so far.
resistance.63,64 This assay works on heminested PCR principle
However, all should have focus upon early diagnosis and
in a closed, completely automated cartridge based system
prompt treatment of every cases of tuberculosis by following
which targets 81 bp fragment of rpoB gene for identification of
guidelines made available by Central Tuberculosis Division,
M TB strain and subsequent probing of this region for muta-
Ministry of Health & Family Welfare.68 The algorithm for
tions that detect Rifampicin resistance.65 An overall sensitivity
diagnosis of pulmonary and extra-pulmonary tuberculosis
of GENEXpert MTB/RIF assay in the diagnosis of pulmonary TB
cases as per Technical and Operational Guidelines for control
was 88%, pooled sensitivity of 98% for smear and culture posi-
of TB in India is shown in Figs. 3 and 4.
tive cases; 68% for smear negative cases.66

Fig. 3 e Diagnostic algorithm for pulmonary TB.68 PLHIV- People Living with HIV, CXR- Chest X-Ray, CBNAAT-Cartridge
Based Nucleic Acid Amplification Test, LPA- Line Probe Assay.
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1 307

Fig. 4 e Diagnostic algorithm for Extra Pulmonary TB.68

The duration of treatment consists of 6e9 months of IP

10. Treatment68 with Kanamycin(Km), Levofloxacin(Lfx), Ethionamide(Eto),
Cycloserine(Cs), Pyrazinamide(z), Ethambutol(E), Isoniazid(H)
RNTCP (now known as National tuberculosis elimination pro- and 18 months of CP with Levofloxacin, Ethionamide, Cyclo-
gramme) has introduced daily regimen for drug sensitive TB in serine, Ethambutol, Isoniazid on daily bases under supervi-
PLHIV, Pediatric TB cases in the entire country and for all TB sion (Table 4).
cases in 104 districts. In drug sensitive TB, for all new TB cases, 8
weeks of intensive phase (IP) with Isoniazid(H), Rifampicin(R), 10.1. XDR TB
Pyrazinamide(Z), Ethambutol(E) in daily doses as per 4 weight
band categories whereas except Pyrazinamide, other 3 drugs All XDR TB cases are treated with injection Capreomycin(Cm),
are continued in continuation phase (CP) for another 16 weeks Moxifloxacin(Mfx), PAS, High dose Isoniazide(H), Clofazimi-
as daily doses. ne(Cfz), Linezolid(Lzd), Co-Amoxyclav for 6e12 months in inten-
For previously treated cases of TB, 12 weeks of IP with sive phase whereas except injectables remaining medications are
Isoniazid(H), Rifampicin(R), Pyrazinamide(Z), Ethambutol(E) continued for 18 months in continuation phase (Table 5).
and injection Streptomycin only for first 8 weeks of IP whereas 20 Since the introduction of PMDT (Programmatic Management
weeks of CP with Isoniazid, Rifampicin and Ethambutol (Table 3). of Drug-resistant TB) in 2007, an increasing trend in diagnosis
MDR/RR-TB cases (with or without additional resistance): and treatment of MDR/RR TB cases was noticed till 2016 as
shown in Fig. 5. In order to decentralize DR TB services (for easy
accessibility, minimize patient travel and maximum patient
Table 3 e Drug regimen for drug sensitive TB (prefix to the
drugs stands for number of months). satisfaction), a total of 628 CBNAAT centers were made opera-
tional by 2016 (Fig. 5).69
Type of Intensive Continuation
As per National Strategic Plan (NSP) 2017e25, following
TB cases Phase(IP) Phase(CP)
have been outlined69
New (2) HRZE (4)HRE
Previously treated (2)HRZESþ(1)HRZE (5)HRE
1. Decentralization of diagnosis and treatment of MDR TB to
The CP may be extended by 12e24 weeks in treating EPTB (i.e. CNS district level
TB, skeletal TB, disseminated TB etc) based on clinical assessment
2. The recently endorsed WHO recommended second line
of treating physician.
probe assay
308 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1

Table 4 e Drug regimen for drug resistant TB (prefix to the drugs stands for number of months) modification of treatment*-
All MDR-TB isolates should be treated with modification in treatment following liquid culture drug sensitivity testing for
Kanamycin and Levofloxacin.
Type of TB cases Intensive Phase(IP) Continuation Phase(CP)
Rifampicin resistant þ Isoniazide sensitive or unknown (6e9)Km Lfx Eto Cs Z E H (18)Lfx EtoCs E H
MDR TB (6e9)Km Lfx Eto Cs Z E (modification of treatment*) (18) Lfx Eto Cs E

Table 5 e Drug regimen for XDR TB (prefix to the drugs stands for number of months).
Type of TB cases Intensive Phase(IP) Continuation Phase(CP)
XDR (6e12) Cm Mfx PAS High dose I Cfz Lzd Amx/clv (18) Mfx PAS High dose I Cfz Lzd Amx/clv

Fig. 5 e Drug resistant TB cases and treatment initiation from 2007 to 2017.69

3. Rapid molecular drug susceptibility test (DST) for second Recommended dose:
line drugs
4. Shorter MDR TB regimen Week 0e2: Bdq 400 mg (4 tablets of 100 mg) daily (7 days per
5. DST guided regimen to cover all variety of DR TB including week) þ optimized background regimen (OBR);
Isoniazide mono-poly DR TB Week 3e24: Bdq 200 mg (2 tablets of 100 mg) 3 times per
6. Use of newer drugs like Bedaquiline week (with at least 48 hours between doses) for a total dose
7. Revised recording reporting system i.e. e-NIKSHAY & of 600 mg per week þ OBR; and
pharmacovigilance systems for active drug safety moni- Week 25 (start of month 7) to end of treatment: Continue
toring & management other second-line anti-TB drugs only as per RNTCP
recommendations.69,70

10.2. Newer drugs

Delamanid (Dlm), nitrpimidazole class of drug which
Bedaquilline (Bdq), diarylquinoline class of drug which targets blocks mycolic acid synthesis and releases nitric oxide upon
ATP synthase thus blocking supply of energy to MTb was metabolism poisoning MTb, has been introduced for the
added to MDR TB regimen from 2015 by WHO. treatment of DR-TB in India (January 2018) after a series of
 i n d i a n j o u r n a l o f t u b e r c u l o s i s 6 7 ( 2 0 2 0 ) 2 9 5 e3 1 1 309

meetings and deliberations with national experts from gov- 2. Sharma SK, Mohan A. Tuberculosis: from an incurable
ernment of India (GoI), WHO country office for India (WHO scourge to a curable disease- journey over a Millennium.
India) and key technical and developmental partners. Indian J Med Res. 2013;137:455e493.
3. Pommerville JC. Alcamo's Fundamentals of Microbiology. 7th ed.
Dosage:
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int/iris/handle/10665/191102.
21. Shirvastva AK, Brahmachari S, Pathak P, Kumar R, Patel U,
The authors have none to declare.
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