A STUDY TO ASSESS THE UTILITY OF THE ADENOSINE DEAMINASE ÀSSAY

(ADA) IN DIFFERENT BODY FLUIDS FOR DIAGNOSIS OF TUBERCULOSIS

AND ITSCOMPARISION WITH THE GENE XPERT MTB/RIF ASSAY

DISSERTATION

SUBMITTED TO

DEPARTMENT OF MICROBIOLOGY

Dr. RAMMANOHAR LOHIA AWADH UNIVESITY, AYODHYA -224202 (UP)

IN PARTIAL FULFILMENT FOR THE DEGREE OF SCIENCES IN

MICROBIOLOGY

BY
Ms. DIVYA PANDEY (M.Sc MICROBIOLOGY)

UNDER THE SUPERVISION OF

DR. RICHA MISHRA

ADDITIONAL PROFFESOR

DEPARTMENT OF MICROBIOLOGY

SANJAY GHANDHI POST GRADUATE INSTITUTE OF MEDICAL SCIENCES

LUCKNOW- 226014UTTAR PRADESH

 CERTIFICATE OF GUIDE

This is to certify that Ms. Divya Pandey a student of M.Sc. Microbiology, at Dr.
Rammanohar Lohia Avadh University Ayodhyahas worked in the Mycobacteriology
laboratory, Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical
Sciences, Lucknow for her dissertation from March to November 2021 on the project
entitled“A study to assess the utility of the Adenosine deaminase Assay (ADA) in
different body fluids for diagnosis of Tuberculosis and its comparison with the Gene
Expert MTB/RIF assay”. All the data has been obtained by the candidate herself and the
data obtained is genuine. She has gained practical experience during this tenure and
successfully completed her dissertation work under the guidance and supervisionof Dr.
Richa Mishra, Additional Professor, Department of Microbiology S.G.P.G.I.M.S.
Lucknow.

Dr. RICHA MISHRA

ADDITIONAL PROFESSOR

DEPARTMENT OF MICROBIOLOGY

SANJAY GANDHI POST GRADUATE INSTITUTE OF MEDICAL SCIENCES,

LUCKNOW-226014,UP
 CERTIFICATE OF HOD

This is to certify that Ms. Divya Pandey a student of M.Sc. Microbiology, Dr. Rammanohar
Lohia Avadh University Ayodhya has worked in the Mycobacteriology laboratory,
Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow for her dissertation from March to November 2021 on the project entitled “A study
to assess the utility of the Adenosine deaminase assay (ADA)in different body fluids for
diagnosis of Tuberculosis and its comparison with the Gene Expert MTB/RIF assay”
under the supervision of Dr. Richa Mishra, Additional Professor, Department of
Microbiology S.G.P.G.I.M.S. Lucknow. All the data obtained by the candidate herself and
the data obtained is genuine.

Dr. UJJALA GHOSHAL

PROFESSOR& HEAD

DEPARTMENT OF MICROBIOLOGY,

SANJAY GANDHI POST GRADUATE INSTITUTE OF MEDICAL SCIENCES,

LUCKNOW-226014,UP
 Dr. Rammanohar Lohia Avadh University, Ayodhya

Centre of Excellence, Department of Microbiology

Dr. Shailendra Kumar Mobile: 9415077035

Professor & Head Email: shailendrakumar@rmlau.ac.in

Date:

CERTIFICATE

This is to certify that dissertation on entitled “A study to assess the utility of the Adenosine
deaminase Assay (ADA) in different body fluids for diagnosis of Tuberculosis and its
comparison with the Gene Expert MTB/RIF assay” Is bonafide work of Ms. Divya
Pandey, student of M.Sc. Microbiology from Dr. Rammanohar Lohia Avadh University
Ayodhya (UP) done under the our joint guidance for a period of six month at S.G.P.G.I.M.S.
Lucknow- 226014 (UP).

(Shailendra Kumar)
 DECLARATION

I, Ms. Divya Pandey certifythat the work embodied in this M.Sc. project is my own bonafide
work carried out by me under the supervision of Dr. Richa Mishra Additional professor
from March to November 2021at Department of Microbiology, Sanjay Gandhi Post
Graduate Institute of Medical Sciences, Lucknow-226014, UP. The matter embodied in
this M.Sc. project has not been submitted for the award of any other degree/diploma.

I declare that I have faithfully acknowledged, given credit to and referred to the research
workers wherever their works have been cited in the text and the body of this project. I
further certify that I have not wilfully lifted up some other’s work, para, text, data, results,
etc., reported in the journals, books, magazines, reports, dissertations, thesis, etc., or available
at web sites and included them in this M.Sc. project and cited as my own work.

DATE: Ms. DIVYA PANDEY

PLACE: (M.Sc. MICROBILOGY)

 ACKNOWLEDGEMENT

This acknowledgment expresses an effort to produce my overwhelming sense of gratitude,

devotion and regards to those helped during my dissertation work. I take this opportunity to
express my deep gratitude and appreciation to those who helped directly and indirectly my
dissertation work.

It is a great pleasure for me to acknowledge my thanks to Dr. Ujjala Ghoshal,Professor &

Head, Department of Microbiology, SGPGIMS, Lucknow, for granting me permission to join
as a dissertation trainee in this Institute.

I do not have words of appreciation and inclination to return kindness of my esteemed mentor
Dr. Richa Mishra, AdditionalProfessor, Department of Microbiology, Lucknow for her
encouragement, devoting her valuable time and ever helping attitude imparted to me from
beginning till the last moment of this work. It is only her blessing; masterly guidance and
constructive criticism from time to time which help me complete the work.

I also take this opportunity to express a deep sense of gratitude to Dr. Akshay Arya, Dr.
Nidhi Pandey and Dr.Mitra Kar, Senior resident, Department of Microbiology for this
ceaseless encouragement, cordial support, valuable information and guidance.

I am thankful to the senior technical officer of the Department of Microbiology for their
continuous support. I am also thankful to Mr. D. K. Chamoli and Mr. D. K. Singh.Their
continuous attention, motivation and deep interest throughout the tenure of my work have
helped me immensely in completing my dissertation work.

I thank Mr. Santosh, Mrs. Jyoti, Mrs. Sadhana Singh, Ms. Shreya Mishra, Mr. A.K.
Pandey and Mr. Raghuveer, Technical staff and entire laboratory staff of Microbiology
Department, who have been a source of constant help and encouragement during my
dissertation.

I am indebted to Dr. Shailendra Kumar, Professor & Head of the Department of

Microbiology, Dr Rammanohar Lohia Avadh University, Ayodhya UP for the all support
throughout my master degree and special thanks to my teachers, Dr Rajeev Gaur, Dr.
Tuhina Verma, Dr. Ranjan Singh, Dr. Manikant Tripathi Dr. Anurag Singh.
At last, I would like to thank all the patients who have unknowingly been the basic
foundation of this dissertation.

Above all, I am thankful to Almighty for making me worthwhile to conduct and complete this
study and for giving me this strength and vigour to proceed in my life.

Ms. DIVYA PANDEY

(M.Sc. MICROBIOLOGY)
 CONTENTS

Sr. No. Title Page No

Introduction
1 1-2

Aims & Objective

2 3

Review of Literature
3 4-18

Material & Methods

4 19-27

Results
5 28-38

Discussion
6 39-41

Conclusion
7 42-42

Bibliography
8 43-51

9 Annexure 52-56
 ABBREVIATION

ADA Adenosine deaminase assay

AFB Acid fast bacilli

CNS Central nervous system

CSF Cerebrospinal fluid

EPTB Extra pulmonary tuberculosis

HIV Human immunodeficiency virus

NTM Nontuberculous Mycobacteria

MTB Mycobacterium tuberculosis

MTBC Mycobacterium tuberculosis complex

PTB Pulmonary tuberculosis

TB Tuberculosis
TBP Tuberculous peritonitis

RIF Rifampicin

SNPTB Smear Negative pulmonary tuberculosis

 INTRODUCTION

Tuberculosis (TB) is a contagious and communicable disease that is the major cause of illness
and one of the top ten causes of mortality worldwide. Until the corona virus (COVID-19)
pandemic, leading cause of death from a single infectious agent (ranking above HIV/ AIDS)
globally in 2020, an estimated 10.0 million (range, 8.9–11.0 million) people became ill with
tuberculosis (TB). There were 1.2 million (range, 1.1–1.3 million) HIV-negative TB deaths,
with an additional 2,08,000 (range, 177 000–242 000) HIV-positive TB deaths (WHO,
Global TB Report;2021). The bacillus, Mycobacterium tuberculosis causes tuberculosis,
which is disseminated by persons who are sick with the disease, coughing and sneezing can
expel bacteria into air. Lungs are the typically affected organs in TB known as Pulmonary TB
but it can also affect any other organs of the body known as extra pulmonary TB (ETPB)
(Pang et. al;2019).Despite an increasing tendency in extra pulmonary case as a percentage to
total tuberculosis cases, the disease is not given significant emphasis on public health agenda
because it is less transmissible than pulmonary tuberculosis (Pang et.al;2019;
Sandgrenet.al;2013).Extra pulmonary tuberculosis is a broad term for a variety of disease that
affect the lymph nodes, pleura, urogenital tract,boneand joints, meninges central nervous
system(CNS),bowel and peritoneum, pericardium and skin (Sharmaet. al;2004).

TB must be diagnosed early in order to receive the proper initial treatment and reduce the
mortality rate (Edwardset. al; 2016). For a successful TB control programme, prompt
diagnosis is critical. Despite the fact that there are numerous methods for diagnosing
tuberculosis, such as Ziehl-Neelsen (Z-N) staining, polymerase chain (PCR), Gene xpert and
culture, these methods lack sufficient sensitivity and specificity in the diagnosis of
tuberculosis infection, ZN staining and culture have a sensitivity of 10-40% and 8-49 %
respectively (Jayet.al ;1985).

Recent studies of populations with high prevalence of tuberculosis report that tuberculous
pleural effusion occurs in approximately 30% of TB patients (Reechaipichitku et.al;
2000).Definitive diagnosis of tuberculous pleural effusion requires demonstration of
Mycobacteriumtuberculosis in the pleural fluid; however the organism is seldom detectable
in the pleural fluid.Conventional methods for diagnosing TB were found to have low
sensitivity and specificity.Similarly, tuberculosis peritonitis (TBP) is one of the most
common extra pulmonary manifestations of tuberculosis, accounting for about 3% of all
EPTB cases (Su et.al; 2019). The reported mortality of this severe type of EPTB ranges from
15% to 31%, which can be related to the non-specific clinical presentation of TBP in the
majority of cases (Talwani et. al; 2000).Unfortunately, conventional laboratory diagnostics
including smear microscopy and mycobacterial culture fail to deliver satisfactory results for
TBP (Michot et.al; 2016). Thus, there is a need for a single test which is adequately sensitive
and specific and at the same time inexpensive and easy to perform (Sharma et.al; 2001).
Several studies have demonstrated the use of ADA in the diagnosis of TB in various fluids
including pleural, meningeal and pericardial (Michot et.al; 2016; Riquelme et.al; 2006; Voigt
et.al; 1989). Because of its simplicity, low cost, and quick results, adenosine deaminase assay
(ADA) was developed and is now widely used to diagnose tuberculosis. ADA's high
sensitivity and specificity (sensitivity of 92 % and specificity of 89 %) for early EPTB
diagnosis have been confirmed in numerous studies.

Adenosine deaminase (ADA) is increased in tuberculous ascitic fluid because of the

stimulation of T cells by the mycobacterial antigens (Hillebrandet.al; 1996).Because of its
simplicity, low cost, and quick results, adenosine deaminase assay (ADA) was developed and
is now widely used to diagnose tuberculosis. ADA's high sensitivity and specificity
(sensitivity of 92 % and specificity of 89 %) for early EPTB diagnosis have been confirmed
in numerous studies. However, the most important biological activity is relegated to the
lymphocyte tissue, as ADA is required for lymphocyte proliferation and differentiation. T
lymphocytes have 10 to 12 times higher ADA levels than B lymphocytes. During
lymphocytic proliferation, the enzyme activity of lymphocytes varies inversely with the
maturity state of lymphocytes(Dwivediet. al; 2006).The activity of adenosine deaminase
(ADA) is measured in the diagnosis of tuberculosis and also in the differential diagnosis of
TB. Adenosine deaminase is a purine catabolism enzyme whose activity has been found to be
increased in a variety of diseases, including tuberculosis, HIV, typhoid, infectious
mononucleosis, and certain cancers, particularly those of the blood. The utility of the ADA
assay in various body fluids in the laboratory diagnosis of extra pulmonary TB (such as
meningeal, pleural, peritoneal, and pericardial TB) and smear negative pulmonary TB
(SNPTB) has been established (Chandar et.al; 2012).

The aim of this study was to assess the utility of the Adenosine Deaminase Assay (ADA)in
different body fluids for diagnosis of Tuberculosisand it’s comparison with the Gene Xpert
MTB/RIF assay.
 AIM AND OBJECTIVE OF THE STUDY

AIM: To assess the utility of the Adenosine deaminase Assay (ADA) in different body fluids
for diagnosis of tuberculosis and its comparison with the Gene Expert MTB/RIF assay

OBJECTIVES:

1. To assess the value of the Adenosine Deaminase Assay in different body fluids for the
diagnosis of tuberculosis

2. To compare the Adenosine Deaminase Assay with the Gene Xpert MTB/RIF
Assay for the diagnosis of tuberculosis
 REVIEW OF LITERATURE

HISTORICAL PERSPECTIVE OF TUBERCULOSIS:

It has been hypothesized that the genus Mycobacterium originated more than 150 million
years ago. Three million year ago an early progenitor of Mycobacterium Tuberculosis might
have infected early hominids in east Africa. Tuberculosis (TB) was called in ancient Greece
as “phthisis”, in ancient Rome as “tabes” and in ancient Hebrew a “Schachepheth”. Because
of the paleness of suffers, tuberculosis was dubbed ‘’the white plague’’ in the 1700s.During
the middle age, Tuberculosis was often referred King Evil or King Touch due to belief that
the king of England and France could cure TB by simply touching affected people (Murray
et.al;2016). A medical papyrus from 1550 BC contains the first documented reference to
tuberculosis in Egypt. A kind of tuberculosis known as Pott’s disease has been discovered
among Egyptian mummies’s spinal tuberculosis. TB has also been identified in India a
Sanskrit hyme (Rig Veda).Dr. Robert Koch reported the discovery of Mycobacterium
Tuberculosis, on 24th March in 1882 (Sakula et.al; 1882). In1890, Koch announced the
discovery of Tuberculin, a substance derived from the tubercle bacilli (Centre for Disease
Control and Prevention).Dr. Robert Koch’s Finding was the most significant step forward in
the fight to control and eradicate this fatal disease. In 1905, he was awarded Noble Prize for
his contributions in the field of TB research. In 1893, Johann Schonlein developed the world
“tuberculosis”. Mycobacterium ulcerans, causing infections since ancient times, requires
specific environmental conditions as reflected nowadays in its distribution worldwide
(Hayman et.al; 1984).The 24th of March, 1982was declared “WORLD TUBERCULOSIS
DAY”.

In 1819, the French Theophile Laennaec identified the presence of consolidation, pleurisy
and pulmonary cavitation as pathognomic signs of pulmonary or extra pulmonary TB
(Danien et.al; 2000).Mycobacterium tuberculosis most commonly affects the respiratory
tract, but it could also infect gastrointestinal, bones, joints, nervous systems, lymph nodes,
genitourinary tract and skin with inflammatory infiltration, caseation, necrosis, abscesses,
fibrosis, formation of tubercles and calcification (Duffin et.al; 1998; Daniel et.al;
2004).Extra-pulmonary phthisic tubercles were recognized in the intestines, liver, meninges
and other organs, as also described by Sir Percivall Pott, a British surgeon that in 1779
defined as "Pott's disease" the vertebral collapse and spinal cord paralysis caused by TB
infection(Daniel et.al; 2004; Dormandy et.al; 1999; Garrison et.al; 1921; Roguin et.al; 2006;
Nuland et.al; 1988).

INFECTIOUS AGENT

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis and commonly

found in human beings. M. tuberculosis, M. bovis, M. africanum, M. microti, and M. canetti
are bacteria from the Mycobacteriumtuberculosiscomplex that are primarily found.
Mycobacterium has over 70 species, two of which are considered major human
pathogens:Mycobacteriumtuberculosis (Tuberculosis or Koch's disease, Koch, 1882) and
Mycobacteriumleprae (leprosy or Hansen's disease, Hansen, 1882). The remaining
Mycobacteriumis environmental in nature and referred to as Mycobacterium Other Than
Tuberculosis (MOTT) organisms. MOTTs cause a variety of opportunistic infections that can
occur without person-to-person transmission and are generally drug resistant. Mycobacterium
avium-intracellulare (MAI) or -complex (MAC), for example, is diagnosed through blood
and/or organ tissue culturing, is common in immunocompromised people, and requires life-
long antibiotic therapy (Abe et. al; 1999).

MORPHOLOGY:

Mycobacteria are typically rod-shaped, non–spore forming, aerobic bacteria, classified as

acid-fast bacilli they do not stain readily and resist to decolourization The dimensions of the
bacilli have been reported to be 1-10 μm in length (usually 3-5 μm), and 0.2 -0.6 μm in width.
Variable morphology can be observed when grown on solid media and some species exist as
shorter cocci-bacilli or curved rods on artificial media (Belislen, et.al; 2010). The reported
morphological variation in M. tuberculosis are classified in two categories; first those which
are frequently seen at exponential phase of growth that is rod, V, Y-shape, branched or buds
second those that are seen occasionally under stress or environmental conditions which are
round, oval, ultra-virus, spore like, and cell wall defiant or L-forms (Velayanti et.al; 2011).
Mycobacteriumtuberculosis is an obligate aerobe. For this reason Mtbcomplex are always
found in the well aerated upper lobes of the lung in patient with TB. The bacterium is
facultative intracellular parasite, usually of macrophages, and has generation time of15-20
hours, which is extremely, slow compared to other bacteria with division times measured in
minutes (E.coli can divide every 20 minute) (Cook et.al; 2009).
CELL WALL:

Cell wall of Mycobacterium bacillus is encompassed by remarkable elaborate cell wall

structure (Smith et.al; 2003). Mycobacteria are non-motile, nonsporulating, acid-fast bacilli
that appear microscopically as straight or slightly curved rods, 1 to 4 μm in length and 0.3 to
0.6 μm wide (Barry et. al; 1996). It is a slow growing mycobacterium with a doubling time of
12–24 h under optimal conditions. A major feature of Mtb is the peculiar cell wall structure,
that provides an exceptionally strong impermeable barrier to noxious compounds and drugs
and that plays a fundamental role in virulence. The remarkable molecular complexity of the
mycobacterial cell wall is a unique feature that distinguishes Mycobacterium species from the
vast majority of other prokaryotes. Although this microorganism is Gram- positive, their
envelopes have some similarities to Gram-negative cell walls, such as an outer permeability
barrier that acts as a pseudo-outer membrane (Minnikin et. al; 1982). Much of the early
structural definition of the cell wall was conducted in the 1960s and 1970s and later
continued by Minnikin, who in 1982 proposed the currently accepted structural model for the
cell wall architecture the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex, as it is
commonly termed, represents the cell wall core structure that encompasses the mycobacterial
bacilli (Adam et. al; 1969).

Fig: 1 cell wall of mycobacteria

EPIDEMIOLOGY OF TUBERCULOSIS

Globally, an estimated 10.0 million (range, 8.9–11.0 million) people fell ill with TB in 2019,
a number that has been declining very slowly in recent years equivalent to 132 cases (range,
118–146) per 1,00,000 population. There were an estimated 1.2 million (range, 1.1–1.3
million) TB deaths among HIV-negative people in 2019 (a reduction from 1.7 million in
2000), and an additional 208 000 deaths (range, 177 000–242 000) among HIV-positive
people (a reduction from 678 000 I 2000). Men (aged ≥15 years) accounted for 56% of the
people who developed TB in 2019; women accounted for 32% and children (aged <15years)
for 12%. Among all thoseaffected8.2% were people living with HIV. Geographically, most
people who developed TB in 2019 were in the WHO regions of South-East Asia (44%),
Africa (25%) and the Western Pacific (18%), with smaller percentages in the Eastern
Mediterranean (8.2%), the Americas (2.9%) and Europe (2.5%). Eight countries accounted
for two thirds of the global total: India (26%), Indonesia (8.5%), China (8.4%), the
Philippines (6.0%), Pakistan (5.7%), Nigeria (4.4%), Bangladesh (3.6%) and South Africa
(3.6%). The other 22 other countries in WHO s list of 30 high TB burden countries
accounted for 21% of the global total.At the end of 2019, the world as a whole, most WHO
regions and many high TB burden countries were not on track to reach the 2020 milestones of
the End TB Strategy. Globally, the TB incidence rate is falling, but not fast enough to reach
the 2020 milestone of a 20% reduction between 2015 and 2020. The cumulative reduction
from 2015 to 2019 was 9% (from 142 to 130 new cases per 100 000 population), including a
reduction of 2.3% between 2018 and 2019. More positively, the WHO European Region has
almost reached the 2020 milestone, with a reduction of 19% in the TB incidence rate between
2015 and 2019, and the African Region has made good progress, with a reduction of 16%. A
total of 78 countries are on track to reach the 2020 milestone, including seven high TB
burden countries that have already reached it (Cambodia, Ethiopia, Kenya, Namibia, the
Russian Federation, South Africa and the United Republic of Tanzania) and three other high
TB burden countries that are on course to do so (Lesotho, Myanmar and Zimbabwe) EPTB
accounted for 15% of the 6.3 million incident cases reported by the WHO in 2016, with rates
ranging from 8% in the WHO Western Pacific Region to 24% in the WHO Eastern
Mediterranean Region (WHO, Global TB Report; 2021).EPTB deserves to be given more
attention in these areas. EPTB patients accounted for 37 percent, 16 percent, and 57.5 percent
of all notified TB cases in previous studies conducted in Oman, India, and Saudi Arabia,
respectively (Gaifer et. al;2017) . The overall number of TB cases decreased by 13%, while
TB meningitis and TB of the bones increased significantly, while TB pleural effusion and
lymphadenopathy remained unchanged (Hoogendorn et.al; 2013). According to a study
conducted in Cameroon in 2010 with 984 patients, about a quarter of patients with PTB also
have EPTB (26.6%),(Peufraet.al; 2013).
EXTRAPULMONARY TUBERCULOSIS

Tuberculosis bacilli spread hematogenously and lymphatically, resulting in extra pulmonary

tuberculosis (EPTB). Protective immunity against the bacteria develops as a result of this
spread and the development of specific cell-mediated immunity mechanisms, such as the
formation of anti TNF alpha, IL12, and interferon gamma, with the formation of encapsulated
granuloma containing viable bacilli. Although this can occur at any time after primary
infection, it is most common years or decades later due to changes in responsible immune
response mechanisms, such as extreme ages (children or the elderly), concurrent medical
conditions, or treatments requiring an immunein extra pulmonary forms of tuberculosis, such
as pleural, meningeal, urinary, peritoneal, and pericardial TB, sensitivity would range from
50 to 70%, with a high specificity of 90 to 95 percent. There is insufficient evidence to
determine the diagnostic yield of these techniques in other locations, though some studies
have reported a sensitivity of around 80% and a specificity of 90% in forms affecting the
bone and lymph nodes (Fesharet.al; 2013).

Tuberculous peritonitis

The clinical presentation may be acute or chronically intermittent. Most patients present with
abdominal pain (80-95%), fever (40-90%), diarrhoea and constipation (11-20%), and weight
loss, anorexia and malaise (40-90%) Patients with peritoneal TB manifest a slowly
progressive abdominal swelling from ascites and abdominal pain. Adhesions may also cause
small intestine obstruction. On physical examination, diffuse abdominal tenderness, doughy
abdomen, hepatomegaly and ascites may also present the risk factors for developing
peritoneal TB are HIV infection, cirrhosis, diabetes, malignancy and receiving continuous
ambulatory peritoneal dialysis (Sanai et.al; 2005).

Miliary Tuberculosis

Military tuberculosis is a type of tuberculosis that affects the in the pathology sample, the
term "miliary" refers to the numerous small pulmonary nodules scattered throughout the lung
like millet seeds. Today, however, it also refers to progressive and widespread forms of
tuberculosis. Meningeal involvement increases mortality, so standard chemotherapy should
be extended from 6 to 9 or 12 months when it is present, and corticosteroids may be helpful
in lowering mortality (Murray et.al; 2016).
Tuberculous Meningitis

The most severe form of EPTB involves the central nervous system (CNS), accounting for 5–
10% of all cases, with TB meningitis (TBM) being the most common complication. TBM is
common in children (under four years of age) and immunocompromised individuals, such as
those with HIV infection, and can occur with or without an associated PTB. In addition to
TBM, CNS disease included intracranial tuberculoma, tuberculous brain abscesses,
arachnoiditis, increased intracranial pressure, and hydrocephalus(Gopalaswamy
et.al;2020Mechai et.al; 2019).

Tuberculous Lymphadenitis

Tuberculous lymphadenitis (TBL), also known as Scrofula, is the most common type of
EPTB, accounting for 35–40% of cases. The disease is usually non-fatal and manifests as
unilateral single or multiple painless lumps, with 60–90 percent of TBL cases affecting the
cervical lymph node. In a few cases, the submandibular and supraclavicular lymph nodes are
involved (Gandhare,et.al; 2017, Gautam, et.al; 2018).

Pleural tuberculosis

One of the most common forms of EPTB is pleural tuberculosis (PLTB), which is associated
with PTB as an immune reaction or miliary TB. PLTB is uncommon in children aged 2 to 12
years old, but it is common in adolescents aged 12 to 16 years old and adults (Chakrabarti,
et.al; 2006, Chiu et.al; 2007, fisher, et.al; 2011).Fever (in about 86 percent of cases), chest
pain, cough, and dyspnoea are the most common symptoms, and it is sometimes accompanied
by loss of appetite, malaise, and weight loss (Shaw, et.al; 2019).

Tuberculous pericarditis

Tuberculous pericarditis is a rare TB manifestation that can be fatal if not diagnosed and
treated properly. TBP is caused by Mtb haematogenous spread and retrograde lymphatic
spread from peritracheal, peribronchial, or mediastinal lymph nodes, as well as primary or
secondary tuberculosis. Pericardial effusion, constrictive pericarditis, and a combination of
effusion and constriction are the three clinical forms of TBP. Fever, weight loss, night sweats,
cough, chest pain, and breathlessness are all symptoms of pericardial effusion, which can
range from mild to severe. Constrictive pericarditis and thick fibrinous fluid around the heart
are seen in the next stage(Mayosi, et.al; 2005, Ntsekhe, et.al; 2013, M.; Tse, et.al; 2015).
Cutaneous Tuberculosis
Common EPTB that affects skin, soft tissues, and musculoskeletal structures (bones and
muscles) are discussed here.1–1.5 percent of all EPTB cases are caused by cutaneous
tuberculosis (CTB).Exogenous, endogenous, or hematogenous spread could all be potential
sources of CTB. There is a direct TB inoculation or tuberculous chancre in an exogenous
spread. The bacilli enter a person who has never been infected with Mtb through minor skin
abrasions or broken skin. The infection appears as a non-tender nodule that enlarges and
erodes into a painless ulcer 2 to 4 weeks after vaccination. When the same thing happens to
someone who already has TB immunity, it's called TB verrucosa cutis, and it looks like a
painful hyperkeratotic or verrucous papule with an inflammatory areola. CTB as a result of
an endogenous source (Barbagallo, et.al; 2002, Charifa, et.al; 2021, Hill, et.al; 2021,Van, et.
al;2015).

PATHOGENESIS:

Pathogenesis of Extra pulmonary TB usually involves all organs of the body except lung
following reactivation via haematogenous spread from primary lung focus or via
hematogeneous spread from active pulmonary or miliary TB, and through lymph channels
from infected lymph nodes or fallopian tubes in women. Both visceral and parietal layers are
affected with the formation of multiple tuberculous nodules (Sanai et.al; 2005).

TRANSMISSION OF TUBERCULOSIS:

Tuberculosis is communicable disease generally spread from person to person through

inhalation of infectious droplets aerosols (1-5) micron in size that is containing tuberculosis
organisms that are expelled into the air via respiratory tract pathways by an infected person
coughing or sneezing. In case of M. bevies, which is spread by cattle and is spread by
consuming infected raw milk,Mycobacterial transmission include occupational inhalation of
droplets during mucus manipulations by laboratory employees or oral tuberculosis infection
in cattle there's no proof that TB is spread through shaking hands, drinking drinks, or
exchanging towels. Coughing, sneezing, spitting, yelling, laughing, chatting, and
expectorating infectious airborne droplets can transmit bacilli to an infected person with
active TB disease, such as pulmonary or laryngeal TB (Mathema et.al; 2006). The bacilli are
inhaled and lodge in the terminal air passages of the lungs, where they proliferate within
macrophages. Bacillary clearance from afflicted tissues or sputum indicates a good host
response (resistance) and cure in response to antibiotic treatment, although any recent
transmissions may complicate MTB infection resolution.

SPREAD OF INFECTION TO OTHER ORGANS:

The mechanisms of spread of tuberculosis to the abdomen are (Sanai, et.al; 2005): -

1. Spread through hematogeneous route from active primary focus in lung.

2. By means of ingestion of infected sputum from active pulmonary focus.

3.Spread from surrounding organs. (Oesophageal spread from mediastinal TB

lymphadenopathy).

4. Spread by lymphatic channels from infected lymph nodes.

5. From Fallopian tubes by retrograde spread to involve peritoneum.

LABOROTARY DIAGNOSIS:

Microbiological laboratory testing includes:

 Microscopy for Acid-fast bacilli (AFB)

 Culture for Mycobacteria
 Molecular tests and
 Adenosine deaminase test for body fluid

MICROSCOPY FOR ACID- FAST BACILLI (AFB) STAIN:

Acid-fast bacilli (AFB) can be detected in tissues and smears using the Ziehl-Neelsen (ZN)
stain. It's easy, inexpensive, and quick. A positive ZN test requires more than 106 bacteria/g
of tissue in the majority of pauci-bacillary EPTB samples, so it has a limited diagnostic value
of 0-40 percent (Purohit et. al; 2007). In the case of pulmonary tuberculosis, centrifugation of
EPTB samples and fluorochrome staining with ultraviolet microscopy improves microscopy
sensitivity by 10% over ZN staining.Furthermore, neither the etiological diagnosis of M.
tuberculosis nor the drug- resistance can be revealed with ZN microscopy (Menzies et. al;
2008).

Conventional methods of staining include:

 Ziehl-Neelsen (ZN) method (hot staining procedure)

Mycobacteria appear as – pink, thin, 2-4 μm long, slender and curved beaded acid-fast bacilli
and colour of background depends on the counterstain used.

Table 1: Interpretation of Mycobacterial Stain

Counter stain Acid-fast Background

Malachite Green Pink Green
Methylene Blue Pink Blue

CULTURE FOR MYCOBACTERIA:

All specimens suspected of containing Mycobacterium spp. were cultured in the laboratory.
Advantages for culture for Mycobacteria are-

 It is a sensitive method for detection of TB and can detect as few as 10 to 100 bacteria
/ml.
 Culture is required for drug susceptibility testing and for species identification.
 For DNA Sequencing and other molecular tests. Many different media have been
developed for M. tuberculosis growth.

The culture media for Mycobacterium spp. are generally classified into two main groups:
solid media (egg- and agar-based) and liquid media. Antibiotics can be added to culture
media in order to prevent the growth of non-specific flora (Ardito,et.al; 2000).
 Table 2: Type of culture media for TB (Martin et.al; 1975)

Solid Media
Egg Based Media Agar based Media Liquid Media
 Löwenstein-Jensen(LJ)  Middlebrook 7H10 Middlebrook 7H9 broth
medium  Middlebrook 7H11 Automated systems
 Ogawa Medium (LJ  Selective 7H11 MGIT 960 system
without Asparagine  (Mitchison’s (BD [Becton,
 Gruft modification ofLJ medium), containing Dickinson and
(containing malachite carbenicillin, Company]
green, penicillin and Amphotericin B, Diagnostic Systems
nalidixic acid as selective Polymyxin B and Cockeysville, Md.)
agents) Trimethoprim as MB/BacTAlert
 Mycobactosel LJ selective agents (bioMérieux,
(containing Durham, N.C.)
malachitegreen,cyclohexi Versa TREK
mide, lincomycin and (formerly ESP
nalidixic acid as selective Culture System II)
agents) (Trek Diagnostic
 Petragnani medium: Systems, Cleveland,
contains twice the Ohio);
concentration of malachite
green (an inhibitor of
contaminating organisms)

SOLID CULTURE METHOD:

EPTB diagnosis is difficult due to the possibility of paucibacillary clinical samples obtained
from inaccessible locations, which reduces the sensitivity of diagnostic tests. Negative results
cannot rule out the presence of TB due to the low sensitivity of conventional smear
microscopy, which ranges from 0% to 40%. Mycobacterial culture yields have been reported
to range from 30% to 80%, but results typically take 2-8 weeks to arrive, which is too long to
aid treatment decisions for Concomitant pulmonary involvement affects about 10% to 50% of
EPTB patients. As a result, all suspected cases of EPTB should be checked for concomitant
PTB to see if the patient is infectious and to help with diagnosis. Despite normal chest
radiography findings, some EPTB patients have positive sputum culture results
(Gopalaswamy, et. al; 2020).

Traditionally, solid culture media for mycobacteria are kept for up to 8 weeks before a
negative result is reported to the physician. Even though it is well documented that liquid
media detect mycobacteria much earlier, most incubation protocols still require a maximum
of 6 weeks (Chihota,et.al;2010) .

LIQUID CULTURE METHOD:

The BacT/ALERT 3D Mycobacterial Detection System employ a colorimetric sensor and

reflected light to monitor the presence and production of carbon dioxide (CO2) dissolved in
the culture medium. Carbon dioxide is produced as the organisms metabolize the substrates in
the medium. Growth of the microorganisms produces CO2, then the colour of sensor at the
bottom of each culture bottle which changes from dark green to light green and then to
yellow. The lighter colour results in an increase in reflectance units as monitored by the
system. Bottle reflectance is monitored and recorded by the instrument every 10 minutes. The
rate and amount of CO2 production is indicative of the rate and amount of growth of the
microorganism (Diaz, et.al; 2000).

MOLECULAR METHOD:

With the purpose of obtaining faster results and early diagnosis of tuberculosis, several
molecular detection methods were introduced.

 Nucleic acid amplification methods

 Post amplification analysis
 Table: 3. Molecular tests used for diagnosis of tuberculosis

Nucleic acid amplification Post amplification analysis

 Amplicor Mycobacterium  Reverse Hybridization/ Line probe

tuberculosis Test (Amplicor) (Roche assays
Diagnostic System Inc. NJ)
 DNA sequencing
 Mycobacterium tuberculosis (MTB)
 Microarray Analysis
Direct Test (MTD) (Gen-Probe Inc.
San Diego, CA)  Strain Typing and DNA
 BD Probe Tec MTB Test (Becton Fingerprinting
Dickinson, Sparks, MD)
 Xpert MTB/RIF assay (Cepheid,
Sunnyvale, CA)
 Real Time PCR
 LAMP (Loop-mediated isothermal
amplification) Assay

The most widely used molecular test which is used for diagnosis of Pulmonary and extra
pulmonary tuberculosis is the Gene Xpert MTB/RIF Assay.

XPERT MTB/RIF ASSAY:

It is a molecular beacon assay for detection of M. tuberculosis and rifampicin resistance

mutations in an 81- bp region (codons 426 to 452) of the rpoB gene known as rifampicin
resistance-determining region (RRDR) (Steingart,et.al;2013). The assay may be used for
AFB smear positive or AFB smear negative samples (direct or concentrated specimens,
spontaneously produced or induced) from adults with suspected TB. Xpert MTB/RIF detects
both live and dead bacteria. The Xpert MTB/RIF assay received endorsement by WHO in
2011 and approved by the FDA in 2013 (Boehme, et.al; 2010).

In 2010, the World Health Organization (WHO) endorsed the roll out of Xpert® MTB/RIF, a
cartridge based automated polymerase chain reaction (PCR) system for detection of MTB
deoxyribonucleic acid (DNA). Xpert provides results in 2 hrs and can detect down to 131
CFU per sample, improving the sensitivity for patients with low bacillary burden compared to
smear microscopy (Steingart,et.al;2013).
The Xpert MTB/RIF automated molecular assay (Cepheid, CA, USA) for rapid diagnosis of
tuberculosis and detection of Rifampicin resistance, a marker of MDR-TB, is a watershed
moment in TB research. The assay was created, optimised, tested, and approved specifically
for sputum-based detection of pulmonary tuberculosis. However, more recently, the assay has
been tested on a variety of extrapulmonary clinical samples. The reported sensitivity of the
EPTB assay is highly variable, ranging from 25% to 96%, but it has consistently exceeded
50% in all but one study. For cerebrospinal, pleural, pericardial, peritoneal, and synovial fluid
samples, however, lower sensitivities have been reported. Its use in the diagnosis of EPTB
will be limited in the coming years due to high costs and lower sensitivity for smear-negative
EPTB samples than for smear-positive EPTB samples (Lawn et.al;2018)

ADENOSINE DEAMINASE ASSAY:

One of the most studied and widely used biomarkers in body fluids for the diagnosis of EPTB
is adenosine deaminase (ADA) activity measurement. ADA is a purine-metabolizing enzyme
found in a variety of tissues, including lymphoid T-lymphocytes.The conversion of adenosine
to inosine is catalysed by the enzyme deaminase (DA). The interaction of the mycobacterium
with host factors causes immune system cell differentiation in humans, which is where ADA
activity comes from. ADA-1 and ADA-2 are two iso-enzymes. Most cells produce ADA-1,
while monocytes and macrophages produce ADA-2 exclusively. In both endemic and
impoverished settings, ADA appears to be a useful test for early TB diagnosis. The NHS
Health Technology Assessment Program conducted a systematic review of ADA tests for the
diagnosis of pulmonary tuberculosis and found that ADA tests were only used in a limited
number of cases. However, there is substantial evidence to support its use for high-sensitivity
EPTB diagnosis. Individual ADA iso-enzymes and their ratios could help distinguish
between the various causes of increased ADA activity in body fluids, particularly in
borderline ADA levels. When compared to other infectious or malignant causes, ADA-2
levels are higher in tuberculosis. In large studies, the use of ADA iso-enzymes in the
diagnosis of EPTB should be investigated further (Dinnes, et.al; 2017).

ADA is involved in the maturation of monocytes into macrophages (Kaya et.al; 2007). It is a
good indicator of active cellular immunity. In the course of an infectious disease
characterised by microorganisms infecting macrophages, it increases in biological fluids. In
humans, for example, an ADA deficiency causes severe lymphopenia and immunodeficiency
(Cimen et.al; 2008). Guisti and Galanti described a spectrophotometric method for measuring
total plasma ADA in 1984. As determined by modifying a Berthelot's reaction, ammonia
(NH3) forms during adenosine conversion, resulting in an intensely blue indophenol with
sodium hypochlorite and phenol in an alkaline solution. As a catalyst, sodium nitroprusside is
used. The absorbance of indophenols measured at a wavelength of 620 nm is directly
proportional to the ammonia concentration. The addition of phenol nitroprusside solution
stops the ADA-catalysed reaction after one hour of incubation at 37oC. Adenosine deaminase
is used for various body fluids, which include:

Pleural effusion

Almost all research workers have shown sensitivity and specificity of 90 percent to 100
percent for the value of ADA in pleural fluid using different cut-off levels of sensitivity and
specificity for diagnosing tuberculous pleural effusion of 100 percent and 94.1 percent,
respectively. 13), which was almost identical to Burgess, et.al;1995(Sensitivity 90 percent
and specificity 89 percent at a cut-off value of 50U/L), (100% sensitivity and 87.5%
specificity at a cut-off value of 53 U/L) 15, and (95.3% sensitivity and 83.33% specificity at a
cut-off value of 40 U/L) (Farhan, et. al; 2013).

Cerebrospinal fluid

When used to distinguish bacterial from tuberculosis meningitis, the yield of ADA may be
low. The ADA value in most assays detected total ADA, which includes ADA-1 and ADA-2,
which could be the explanation. As a result, fluid with a high cell count (e.g., bacterial
meningitis) can have a high total ADA and be difficult to distinguish from tuberculous
meningitis.The ability of ADA activity in the CSF of HIV-positive patients to diagnose
tuberculous meningitis was limited.

Ascitic fluid

The most common finding is ascites, which is present in about 78 percent of patients with
tuberculous peritonitis. The stimulation of T cells by mycobacterial antigens causes an
increase in ascites ADA in tuberculous ascitic fluid. The presence of ADA in the peritoneal
fluid has been shown to be a simple and reliable method for detecting tuberculous peritonitis
early (Martimez, et.al; 1986).There has been reports of sensitivity and specificity levels
exceeding 90%.
Pericardial fluid

A prospective study in South Africa found that an ADA cut-off level of 40 U/l resulted in test
sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic
efficiency of 84 percent, 80 percent, 91 percent, 66 percent, and 83 percent, respectively. The
researchers looked at pericardial fluid ADA levels as well as histopathology of pericardial
biopsy and discovered a cut-off ADA level of 40 U/L in pericardial fluid(Mathur, et.al;2006).
 MATERIAL AND METHODS

Study setting: This study was conducted in the Mycobacteriology section of the Department
of Microbiology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, a
tertiary care centre in northern India.

Duration of study: March 2021 to November 2021

Study design: A laboratory based prospective study

Sample size:A total of 261body fluids from clinically suspected cases of tuberculosis,
attending outpatient clinics or admitted in various clinical departments at SGPGIMS were
included in the study.

Sample collection:In a sterile container or syringe pleural fluid, ascitic fluid, cerebrospinal
fluid, pericardial fluid and other body fluids samples were collected from patients clinically
suspected fortuberculosis.

Microbiological laboratory analysis: All samples handling and processing was done in a
Class II, A2 type Biosafety cabinet (BSC).

The following tests were performed in our laboratory:

ZIEHL-NEELSEN (ZN) STAIN

PROCEDURE

1. The slides were fixed by flaming.

2. The slides were placed on the staining rack so they were at least 1 cm apart, and flooded
with Carbol fuchsin.

3. The slides were heated by flaming underneath with a methylated spirit-soaked torch, until
fumes appear for 5 to 10 minutes. The stain was not allowed to boil or dry. Additional stain
was added if necessary.

4. Distilled water was added to wash off the stain.

5. The slides were flooded with 5% acid-alcohol.

6. The flooded slides were allowed to stand for 2-3 min.

7. The acid-alcohol was washed off with distilled water.

8. The slides were flooded with 25% H2SO4 and allowed to stand for 2-3 minutes and
washed with distilled water.

9. The slides were flooded with malachite green and allowed to stand for 1-2 minute.

10. The malachite green was washed off with distilled water.

11. The slides were tilted on the rack to dry.

12. Using a microscope, Ziehl-Neelsen smears were screened under 40X and the findings
were confirmed with the 100X oil objective (10X eye piece for a total of 1000X
magnification).

Fig: 2 Growth of mycobacteria in Lowenstein Jensen (LJ) Media

PROCEDURE

1. 3-5 ml sample was homogenized for 15 minutes in a shaker using equal volumes of
4% sodium hydroxide.
2. After centrifugation at 3000 rpm for 15 minutes, the deposit was neutralized with 15
ml of sterile distilled water.
3. The samples were again centrifuged and each slope was inoculated using the
sediment.
4. The cultures were incubated at 35 C to 37 C. All slopes were observed for
occurrence of growth at weekly intervals for eight weeks.
5. The inoculated slopes were examined for growth every week up to eight weeks. The
absence of growth at the end eighth week was regarded as negative culture.

6. Cultures were read twice in the first week, followed by once a week for up to 8 weeks.
Typical non-pigmented, rough, dry colonies were observed on LJ media.

ADENOSINE DEAMINASE ASSAY:

The following materials were arranged, labelled and brought to room temperature before
starting the test:

 Sample reagents

 Both the Phenol reagent & Hypochlorite reagent were diluted 1:5 with distilled water
before use (1 part of reagent + 4 parts of distilled water). The working phenol reagent and
working hypochlorite reagent remain stable for at least 6 months when stored at 2 C-8 C
in tightly closed bottles.

 Sterile 20μL and 1 ml micropipette and pipette tips

 Glass test tubes (no. 16 X 100)

 Water bath/ incubator (35 C - 37 C)

 Spectrophotometer with filter at 570-630 nm at 37 C The ascitic and PD Fluid samples

were reported to have stable ADA values for 2 days at 2 C-8 C, as after this, ammonia
may be released from the samples even without any microbial contamination.

Fig: 3 Adenosine deaminase activity test

PROCEDURE

Pre-requisites of performing the test:

1. All reagents and samples were brought to room temperature before use.
2. The working phenol reagent and working hypochlorite reagent was prepared
beforehand in the same dilution as mentioned above.
3. To measure the Absorbance, the spectrophotometer filter was set at 570-630 nm (Hg
578 or 623 nm) at 35 C- 37 C.
4. The clean dry test tubes were labelled Blank (B), Standard (S), Sample Blank (SB)
and Test (T).
Into the test tube labelled B, 0.20 ml of buffer reagent and 0.02 ml of deionised water was
added.

Into the test tube labelled S, 0.20 ml of buffer reagent and 0.02 ml of standard was added.

Into the test tube labelled SB, only 0.20 ml of Adenosine Reagent was added.

Into the test tube labelled T, 0.20 ml of Adenosine Reagent and 0.02 ml of centrifuged
sample was added

The contents of each tube were mixed well and after covering the top of the tubes with
aluminium foil, they were incubated at 37⁰ C for exactly 60 minutes

After completing incubation, to each tube 1 ml of Working phenol reagent and 1 ml of

Working hypochlorite reagent were added. 0.02 ml of sample was added to SB tube
only.

The reagents were mixed and incubated at 37⁰ C for 15 minutes or at RT for 30 minutes

The Absorbance of the Blank (Abs B), Standard (Abs S), Sample Blank (Abs SB) and
Test (Abs T) against distilled water was measured
CALCULATION

ABSORBANCE TEST- ABSORBANCE STANDARD BLANK

x 50
ABSORBANCE STANDARD – ABSORBANCE BLANK

INTERPRETATION

For serum, plasma, pleural, pericardial, peritoneal, ascitic fluid and body fluid

Serum, Plasma, Pleural, Normal < 30 U/L

Pericardial and Ascitic Suspect 30U/L to 40U/L
Strong suspect 40 U/L to 60 U/L
Positive > 60U/L
CSF Normal <10U/L
Positive >10U/L

GENE XPERT MTB/RIF ASSAY:

GeneXpert Instrument System (catalog number varies by configuration): 6-color GeneXpert

instrument, computer with proprietary GeneXpert Software Version 4.3 or higher, barcode
scanner, and operator manual.

 Printer: If a printer is required, contact Cepheid Technical support to arrange for the
purchase of a recommended printer.
 Leak-proof, sterile, screw-capped specimen collection containers
 Disposable gloves, eye protection, laboratory coats, and labels or permanent marking
pen
 Sterile, single use pipettes with dry-end barrier plugs for sample processing
 Timer
 Fig: 4 Gene Xpert MTB/RIF Assay

Pre-requisites of performing the test:

The following materials were arranged, labelled and brought to room temperature before
starting the test:

 Xpert MTB/RIF cartridge

 Sample reagent (provided in Xpert MTB/RIF kit)

 Sterile (individually packed) disposable transfer pipettes- with single mark for minimum
volume of sample transfer to cartridge (provided in Xpert MTB/RIF kit)
 A plastic disposable pipette was used to collect minimum 1 ml of sample in a sterile
15 ml centrifuge tube.

Using separate plastic disposable pipette, sample reagent at 2:1 (v/v) ratio to sample
was added to the centrifuge tube.

The centrifuge tube was screw capped and closed tightly and then shaken
vigorously 10- 20 times using back and forth movements in a single shake

It was then incubated for 15 minutes at room temperature.

2 ml of the liquefied sample was transferred into the open port of the Xpert MTB/RIF
cartridge using a sterile transfer pipette

The cartridge was then loaded in the instrument.

Interpretation of results:

The results were interpreted by the GeneXpert DX Systems from measured fluorescent
signals and embedded calculation algorithm and were displayed in the “View Results”
window. The results were either MTBc Not Detected or MTBc Detected.

The MTBc detected results were displayed as shown in Table depending on the Ct value of
the MTBc target DNA present in the sample.
 Table:4MTBc detection and co-relation with Ct value

MTB result Ct range

High <16
Medium 16-22
Low 22-28
Very low >28

In those samples in which MTBc was detected, additional interpretation of rifampicin

resistance was as follows:

Table: 5Interpretation of results of rifampicin resistance

Result
Rifampicin Resistance DETECTED
Rifampicin Resistance INDETERMINATE
Rifampicin Resistance NOT DETECTED
Interpretation
A mutation in the rpoB gene has been
detected that falls within the valid delta Ct
setting.
The MTB concentration was very low and
resistance could not be determined.
No mutation in the rpoB gene has been
detected
 RESULTS AND OBSERVATIONS

The study was conducted in 1609 bedded tertiary care centre from March to November 2021,
261 body fluid samples from clinically suspected patients of Pulmonary and extra pulmonary
tuberculosis were included in our study. From each patient, a single non-repeat sample of
body fluids which include pleural fluid, ascitic fluid, cerebrospinal fluid, pericardial fluid,
drain fluid, percutaneous transhepatic biliary drain fluid and synovial fluid were included in
our study.

11, 4% 35, 14%

128, 49%
13, 5%

74, 28%

Pleural fluid Ascitic fluid Cerebrospinal fluid

Pericardial fluid other body fluids

Fig: 1 Distribution of samples include in the study (n=261)

The majority of our samples were pleural fluid (128, 49%) samples.

Fig 1.Shows the distribution of samples included in our study, out of 261, 128 (49%) were
pleural fluid samples followed by 74 (28%) ascitic fluid samples, 13 (5%) cerebrospinal fluid
samples, 11 (4%) pericardial fluid samples and 35 (14%) other body fluid samples.
The 261 samples included in our study were obtained from the outpatient department (OPD)
or inpatient department (IPD) of various clinical specialities of our institute. Fig 2.shows the
distribution of samples obtained from various clinical departments of our institute

41, 16%
66, 25%
12, 5%

15, 6%

52, 20%
37, 14%

38, 14%

Gastroenterology Pulmonary medicine Emergency medicine

Nephrology Critical care medicine Haematology
Other

Fig:2 Distribution of samples obtained from various clinical departments of the institute
(n=261)

As shown in Fig 2, out of 261, 66 (25%) samples were obtained from the Department of
Gastroenterology (41, 55. 40% ascitic fluids; 10, 7.8% pleural fluids and 4, 36.36%
pericardial fluids) , 52 (20%) samples from the Department of Pulmonary medicine (49,
38.28% pleural fluids and 1, 1.35% ascitic fluid), Department of Emergency medicine (n=
38) (15, 11.71% pleural fluids, 15, 20.27% ascitic fluid and 2, 18.18% pericardial fluid)and
Department of Nephrology (n=37) (26,20.31% pleural fluids, 5, 6.75% ascitic fluids and 4,
30.76% cerebrospinal fluid ).
Fig 3.represents the number of patients included in various age groups. The mean age of the
patients included in the study, irrespective of gender was 45.07 ± 18.45 years. The maximum
number of patients included in our study were in the 51-60 years age-group, (n= 54).

60
54
51
Number of patients included in the study

50
42
40
34
32
30

19
20
12
10
10
5
2
0
≤ 10 11 to 20 21 to 30 31 to 40 41 to 50 51 to 60 61 to 70 71 to 80 81 to 90 91 to 100
Age group (in years)

Fig: 3Age distribution of all patients included in the study (n=261)

Among the 261 patients included in the study, 193 patients (74%) were males and 68 (26%)
were females. Fig 4. shows the gender distribution of all patients included in the study

68, 26%

193, 74%

Males Females

Fig: 4 Gender wise distribution of all patients included in the study (n=261)
PLEURAL FLUID:

The ADA values of the pleural fluids included in the study ranges from 2.0 U/L to 264.4 U/L.
Table 1.represents the interpretation of ADA values among the 128 (49%) pleural fluid
samples included in the study. The mean ADA value of these pleural fluid samples was 52.34
± 50.69U/L.

Table: 1The interpretation of ADA values among pleural fluid samples (n= 128)

ADA interpretation ADA values No. of samples in each category n, %

Normal < 30 U/L 59 (46.09%)

Suspect 30U/L to 40U/L 14 (10.93%)

Strong suspect 40 U/L to 60 U/L 17 (13.28%)

Positive > 60U/L 38 (29.68%)

PLEURAL FLUID

38, 29.68%
59, 46.09%

14, 13.28%

14, 10.93%
<30 U/L 30 U/L to 40 U/L 40 U/L to 60 U/L >60 U/L

Fig: 5 Distribution of Adenosine deaminase values in pleural fluid samples (n=128)

The pie chart (Fig 5.) shows the distribution of ADA values in pleural fluid samples. Majority
of the pleural fluid samples included in our study have an ADA value of < 30U/L (59,
46.09%). The number of ADA positive pleural fluid samples in our study, with an ADA
value >60U/L were 38 (29.68%).

Out of the 128 pleural fluid samples included in the study, only 8 (6.25%) pleural fluid
samples were positive by Gene Xpert MTB/RIF assay. Out of these 8 samples, only 7
samples were ADA positive.Only one sample of pleural fluid was positive by culture.

Table: 2 Comparison of ADA positive pleural fluid samples with Gene Xpert MTB/RIF
Assay (n=38)

ADA positive Xpert positive Xpert negative Total

(ADA> 60U/L)
38 7 (18.42%) 31(81.57%) 38
As shown in Table 2, Out of 128 pleural fluid samples, a total of 38 (29.68%) were ADA
positive i.e. they had an ADA value > 60 U/L. Out of the 38 samples, 7 (18.42%) samples
were Gene Xpert positive, and 31 (81.57%) were Gene Xpert negative. One pleural fluid
sample with an ADA value below 30 U/L was positive by Gene Xpert MTB/RIF assay.

ASCITIC FLUID:

The ADA values of the ascitic fluids included in the study ranges from 2.3 U/L to 148.0 U/L.
Table 3. represents the interpretation of ADA values among the 74 (28%) ascitic fluid
samples included in the study. The mean ADA value of these ascitic fluid samples was 28.25
± 31.64 U/L.

Table: 3The interpretation of ADA values among ascitic fluid samples (n= 74)

ADA interpretation ADA values No. of samples in each category n, %

Normal < 30 U/L 53 (71.62%)

Suspect 30U/L to 40U/L 9 (12.16%)

Strong suspect 40 U/L to 60 U/L 3 (4.05%)

Positive > 60U/L 9 (12.16%)

 ASCITIC FLUID
9, 12.16%

3, 4.05%

9, 12.16%
53, 71.62%

<30 U/L 30 U/L to 40 U/L 40 U/L to 60 U/L >60 U/L

Fig: 6 Distribution of Adenosine deaminase values in ascitic fluid samples (n=74)

The pie chart (Fig 6.) shows the distribution of ADA values in ascitic fluid samples. Majority
of the ascitic fluid samples included in our study have an ADA value of < 30U/L (53,
71.62%). The number of ADA positive ascitic fluid samples in our study, with an ADA value
>60U/L were 9 (12.16%).

Out of the 74 ascitic fluid samples included in the study, none of the samples were positive
by Gene Xpert MTB/RIF assay.

Table: 4 Comparison of ADA positive ascitic fluid samples with Gene Xpert MTB/RIF
Assay (n=9)

ADA positive Xpert positive Xpert negative Total

(ADA> 60U/L)
9 0 (0%) 9 (100%) 9
As shown in Table 4, Out of 74 ascitic fluid samples, a total of 9 (12.16%) were ADA
positive i.e. they had an ADA value > 60 U/L. Out of the 9 samples, none of the samples
were positive by Gene Xpert MTB/RIF assay.

PERICARDIAL FLUID:

The ADA values of the pericardial fluids included in the study ranges from 20.40 U/L to
147.90 U/L. Table 5.represents the interpretation of ADA values among the 11 (4%)
pericardial fluid samples included in the study. The mean ADA value of these pericardial
fluid samples was 62.88 ± 44.65U/L.

Table: 5The interpretation of ADA values among pericardial fluid samples (n=11)

ADA interpretation ADA values No. of samples in each category n, %

Normal < 30 U/L 3 (27.27%)

Suspect 30U/L to 40U/L 0 (0%)

Strong suspect 40 U/L to 60 U/L 4 (36.36%)

Positive > 60U/L 4 (36.36%)

 PERICARDIAL FLUID

3, 27.27%
4, 36.36%

4, 36.36%

<30 U/L 30 U/L to 40 U/L 40 U/L to 60 U/L >60 U/L

Fig: 7 Distribution of Adenosine deaminase values in pericardial fluid samples (n=11)

The pie chart (Fig 7.) shows the distribution of ADA values in pericardial fluid samples. The
ADA values of pericardial fluid samples included in our study ranges from 40 U/L to >60
U/L (8, 72.73%). The number of ADA positive pericardial fluid samples in our study, with an
ADA value >60U/L were 4 (36.36%).

Out of the 11 pericardial fluid samples included in the study, none of samples were positive
by Gene Xpert MTB/RIF assay.

Table: 6.Comparison of ADA positive pericardial fluid samples with Gene Xpert
MTB/RIF Assay (n=11)

ADA positive Xpert positive Xpert negative Total

(ADA> 60U/L)
4 0 (0%) 4 (100%) 4

As shown in Table 6, Out of 11 pericardial fluid samples, a total of 4 (36.36%) were ADA
positive i.e. they had an ADA value > 60 U/L. Out of the 4 samples, none of the samples
were positive by Gene Xpert MTB/RIF assay.
CEREBROSPINAL FLUID:

The ADA values of the cerebrospinal fluids included in the study ranges from 1.0 U/L to 15.8
U/L. Table 7.represents the interpretation of ADA values among the 13 (5%) cerebrospinal
fluid samples included in the study. The mean ADA value of these cerebrospinal fluid
samples was 5.5 ± 4.89U/L.

Table: 7 The interpretation of ADA values among cerebrospinal fluid samples (n=13)

ADA interpretation ADA values No. of samples in each category n, %

Normal < 10 U/L 11 (84.61%)

Positive >10U/L 2 (15.38%)

CEREBROSPINAL FLUID
2, 15.38%

11, 84.61%

0-10U/L >10U/L

Fig 8. Distribution of Adenosine deaminase values in cerebrospinal fluid samples (n=13)

The pie chart (Fig 8.) shows the distribution of ADA values in cerebrospinal fluid samples.
Majority of the cerebrospinal fluid samples included in our study have an ADA value of
<10U/L (11, 84.61%). The number of ADA positive cerebrospinal fluid samples in our study,
with an ADA value >10U/L were 2 (15.38%).
Out of the 13 cerebrospinal fluid samples included in the study, none of samples were
positive by Gene Xpert MTB/RIF assay.

Table 8.Comparison of ADA positive cerebrospinal fluid samples with Gene Xpert
MTB/RIF Assay (n=2)

ADA positive Xpert positive Xpert negative Total

(ADA> 10U/L)
2 0 (0%) 2 (100%) 2

As shown in Table 8, Out of 13 cerebrospinal fluid samples, a total of 2 (15.38%) were ADA
positive i.e. they had an ADA value > 10 U/L. Out of the 2 samples, none of the samples
were positive by Gene Xpert MTB/RIF assay.

OTHER BODY FLUIDS:

The ADA values of other body fluids included in the study ranges from 2.10 U/L to 186.50
U/L. Table 9.represents the interpretation of ADA values among the 35 (14%) other body
fluid samples included in the study. The mean ADA value of these body fluid samples was
39.56 ± 42.96U/L.

Table: 9The interpretation of ADA values among body fluid samples (n=35)

ADA interpretation ADA values No. of samples in each category n, %

Normal < 30 U/L 21 (60%)

Suspect 30U/L to 40U/L 1 (2.85%)

Strong suspect 40 U/L to 60 U/L 7 (20%)

Positive > 60U/L 6 (17.14%)

 OTHER BODY FLUIDS

6, 17%

7, 20%

21, 60%

1, 3%

<30 U/L 30 U/L to 40 U/L 40 U/L to 60 U/L >60 U/L

Fig: 9 Distribution of Adenosine deaminase values in other body fluids samples (n=35)

The pie chart (Fig 9.) shows the distribution of ADA values in body fluid samples. The
majority of sample of body fluid samples included in our study have an ADA value of <
30U/L (21, 60%). The number of ADA positive body fluid samples in our study, with an
ADA value >60U/L were 6 (17.14%).

Out of the 35 body fluid samples included in the study, none of samples were positive by
Gene Xpert MTB/RIF assay.

Table: 10Comparison of ADA positive body fluids samples with Gene Xpert MTB/RIF
Assay (n=35)

ADA positive Xpert positive Xpert negative Total

(ADA> 60U/L)
6 0 (0%) 6 (100%) 6

As shown in Table 10, Out of 35 fluid samples, a total of 6 (17.14%) were ADA positive i.e.
they had an ADA value > 60 U/L. Out of the 6 samples, none of the samples were positive by
Gene Xpert MTB/RIF assay.
 DISCUSSION

We aimed to assess the utility of the Adenosine deaminase Assay (ADA) in different body
fluids for the diagnosis of tuberculosis and its comparison with the Gene Expert MTB/RIF
assay in our study. As India is a TB-endemic country, a considerable number of TB patients
present with tuberculous pleuritis, peritonitis or meningitis. Due to lack of appropriate
laboratory methods, the diagnosis of these cases largely depends on clinical suspicion and
non-microbiological parameters. Consequently, the cases having atypical clinical presentation
remain undiagnosed and even untreated. Demonstration of AFB, culture, CT and MRI scan
etc. are the various means to confirm the etiology of tuberculosis but visualization of AFB in
direct smears or in cultures is usually difficult (Molavi et.al; 1985) and in most cases
negative. Newer methods such as those involving the amplification of bacterial DNA by the
PCR and comparable systems, are not available for widespread use in the developing
countries. Routine laboratory findings may not be helpful to differentiate tuberculous etiology
from other causes. In a cross sectional study by Shadai et al, out of 53 suspected TB samples
only 18.7% revealed smear and culture positivity (Shadai et.al; 2015). The poor sensitivity of
AFB smear and culture has widely been reported in many other studies (Ahmed et.al; 2011;
Sharma et.al; 2004; Bueno et.al; 1990; Mrinal et.al; 2014).

Cell-mediated response is the predominant form of immune response to tuberculous

infection, while both cell-mediated and humoral immune responses are elicited by most non-
tuberculous infections in human body. Thus ADA activity, a marker of T-cell activation and
cell-mediated immune response can help differentiate tubercular etiology from non-
tubercular (Gupta et.al; 2010). In TB endemic countries like India, patients with raised ADA
levels could be a signal for tuberculosis where AFB positivity is low. This may help in early
diagnosis and institution of ATT, thus can significantly alter not only the spread but also the
progress and prognosis of TB in our country. Our study is a unique study debating the
significance of ADA estimation in diverse form of extra-pulmonary TB including
musculoskeletal tuberculosis. ADA estimation can also be of considerable help in western
countries where other causes of lymphocyte rich effusions should be considered like
coccidiodomycosis and histoplasmosis. ADA levels, in these cases, are found to be below the
cut-off value and thus save the patient from unnecessary anti-tubercular treatment (Gupta
et.al; 2010). In this study, the utility of the Adenosine deaminase assay in different body
fluids for the diagnosis of tuberculosis and its comparison with the Gene Xpert MTB/RIF
assay was assessed.
We documented male predominance (193:68) among the patients with suspected tuberculosis
in our study. However, other authors have not reported any gender predominance in their
studies. The median age of all patients (n=261) in our study was 47 years. This is similar to
the age distribution reported in other studies.

In our study, we have assessed the diagnostic value of ADA in samples of pulmonary TB and
extrapulmonary TB [TB meningitis (CSF sample), synovial fluid, pleural fluid, ascitic fluid,
abdominal fluid] patients. Smears of body fluid specimens for acid-fast bacilli are insensitive
and culture for M. tuberculosis takes longer time to confirm the diagnosis. In our study only
one pleural fluid sample was positive by culture, while none of the samples were positive on
AFB smear. In our study, the mean ADA value of all the pleural fluids (128, 49%) for
diagnosis of tuberculous pleuritis was 52.34 ± 50.69U/L, and 38 (29.68%) pleural fluid
samples were ADA positive (> 60 U/L). Similar findings were observed in a study by Piras
et.al which was first to report high ADA in tubercular pleural effusion (Piras et.al; 1978).
Subsequently several workers explored its efficacy in the diagnosis of tuberculosis (Ocana
et.al; 1983) and determined that pleural fluid ADA level less than 40 U/L virtually excludes
the diagnosis of tuberculosis (Aggarwal et.al; 1999). Out of the 38 pleural fluid samples that
were ADA positive (ADA > 60 U/L), 7 (18.42%) were positive by Gene Xpert MTB/RIF
assay, as represented in Table 2. All the 7 pleural fluid samples were sensitive to Rifampicin.
Only one pleural fluid with ADA negative (ADA < 30 U/L) was Xpert positive and
Rifampicin sensitive. The mean ADA value of all the ascitic fluid samples (74, 28.35%) for
the diagnosis of tuberculous peritonitis was 28.25 ± 31.64 U/L and 9 (12.16%) ascitic fluid
samples were ADA positive (> 60 U/L). In a study conducted by Lahore et.al, mean (SD)
adenosine deaminase (ADA) level in tuberculosis ascitic fluid was 66.3 ± 30 and that of non-
tuberculosis ascites was 14.2 ± 6.31. Similar high value of adenosine deaminase was also
observed in other studies (Dwivedi et.al;1990 ; Sathar et.al;1995 ;Voigt et.al;1989 ;Sharma
et.al;2016 ). None of the ascitic fluid samples that were ADA positive were positive by Gene
Xpert MTB/RIF assay as represented in Table 4. The mean ADA value of all the pericardial
fluid samples (11, 4%) for the diagnosis of tuberculous pericarditis was 62.88 ± 44.65U/L
and 4(36.36%) pericardial fluid samples were ADA positive (> 60 U/L), similar findings
were observed in a study by Dogan et.al; 1999. None of the pericardial fluid samples that
were ADA positive were positive by Gene Xpert MTB/RIF assay as represented in Table 6.
The mean ADA value of all the cerebrospinal fluid samples (13, 5%) for the diagnosis of
tuberculous meningitis was 5.5 ± 4.89U/L and 2 (15.38%) cerebrospinal fluid samples were
ADA positive (> 10 U/L). In a similar study, significantly higher CSF ADA activity (ADA >
5 U/L) has been observed in confirmed TBM as compared to clinical TBM cases
(Selvakumar et.al; 1991). None of the cerebrospinal fluid samples that were ADA positive
were positive by Gene Xpert MTB/RIF assay as represented in Table 8. The other body fluid
samples included in our study were drain fluids, synovial fluids and percutaneous
transhepatic biliary drain fluids (PTBD). The mean ADA value of all the other body fluid
samples (35, 14%) for the diagnosis of tuberculosis was 39.56 ± 42.96U/L and 6 (17.14%)
body fluid samples were ADA positive (> 60 U/L). None of these body fluids were positive
by Gene Xpert MTB/RIF assay as represented in Table 10.

In conclusion, the use of ADA test in diagnosing PTB or EPTB can be used for diagnosis in
the light of the patient's clinical condition. ADA test can be conveniently performed in many
health care centre with inadequate diagnostic amenities other than mycobacterial culture,
PCR etc. In addition, it is affordable and it has good sensitivity in diagnosing both PTB and
EPTB. ADA may be used for early diagnosis of TB, especially in case of negative AFB
smear from the body fluid specimens. However, culture remains the gold standard for the
confirmatory diagnosis. Prompt treatment of TB is crucial, in areas with high burden of TB.
Further research regarding ADA is necessary to improve specificity, minimize false positive
and choose the suitable cut-off value for specific samples.
 SUMMARY AND CONCLUSION

 This study was conducted at a 1609 bedded, tertiary care hospital in northern India
over a period of 6 months in the Mycobacteriology section of the Department of
Microbiology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow.

 In this study, we assessed the utility of the Adenosine deaminase Assay (ADA) in
different body fluids for diagnosis of Tuberculosis and its comparison with the Gene
Expert MTB/RIF assay, in 261 clinically suspected cases of tuberculosis.

 Out of 261 samples processed in this study, 128 (49.04%) were pleural fluid samples,
74 (28.35%) were ascitic fluid samples, 13 (5%) cerebrospinal fluid samples, 11 (4%)
pericardial fluid samples and 35 (14%) other body fluid samples (which included
synovial fluid, drain fluid and percutaneous transhepatic biliary drain fluid). For each
sample, Adenosine deaminase test and Gene Xpert MTB/RIF assay were performed.

 Adenosine deaminase test was performed for all 261 samples.

 The Gene Xpert MTB/RIF assay was performed for samples.

 None of the samples were positive for acid-fast bacillion ZN stain and only one
pleural fluid sample was positive by culture.

 Out of 128 pleural fluid samples, 38 (29.68%) were ADA positive (> 60 U/L), out of
38 ADA positive pleural fluid samples 8 (6.25%) were Xpert MTB/RIF Assay
positive, out of which 7 were ADA positive whereas 1 was ADA negative.

 Eight pleural fluid samples were positive for MTB complex by Xpert MTB/RIF
assay, all of them were sensitive to Rifampicin and one sample was positive by
culture.

 Out of 74 ascitic fluid samples, 9 (12.16%) were ADA positive (>60 U/L), none of
the samples were positive by Xpert MTB/RIF assay or culture.
 Out of 11 pericardial fluid samples, 4 (36.36%) were ADA positive (>60 U/L), none
of the samples were positive by Xpert MTB/RIF assay or culture.

 Out of 13 cerebrospinal fluid samples, 2 (15.38%) were ADA positive (>60 U/L),
none of the samples were positive by Xpert MTB/RIF assay or culture.

 Out of 35 body fluid samples, 6 (17.14%) were ADA positive (>60 U/L), none of the
samples were positive by Xpert MTB/RIF assay or culture.

The results of our study are important in the early diagnosis of tuberculosis because
pulmonary and extrapulmonary tuberculosis may have an insidious onset and non-specific
clinical presentation; in case of tuberculous pleuritis, peritonitis and meningitis it poses a
a major diagnostic challenge which leads to a delay in diagnosis and even death. So, early
and affordable diagnosis of tuberculosis can be made using ADA at hospitals and health
care centres where advanced infrastructure and laboratory facilities are not available.
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 ANNEXURE 1

ZN STAIN PREPRATION:

Preparation of 1% Carbol fuchsin:

1. Using a digital balance weigh out 1 g of Basic fuchsin in a sterile 100 ml flask.

2. Add 100 ml of absolute alcohol and dissolve the dye by placing it in a water bath at 60° C.
Avoid direct heating (Solution 1).

3. using a digital balance weigh 5 gm of phenol crystals in a balance and transfer it to 100 ml
Erlenmeyer flask

4. Add 50 ml of distilled water and dissolve the phenol crystal. Gentle heat may be required.
(Solution 2)

5. Mix solution 1 and solution 2 and transfer the contents into a 100 ml measuring cylinder.

6. Add distilled water to make up the final volume to 100 ml.

7. Pour the solution through filter paper (what man No 1) and store filtered solution in an
amber colored bottle.

8. Label bottle with name of reagent (1% Carbol Fuchsin), lot number, date of preparation,
expiration date & storage temperature.

9. Tighten caps & store at room temperature.

Preparation of 25% Sulphuric acid:

1. Using a 100 ml measuring cylinder measure out 75 ml of distilled water in 500 ml conical
flask and submerged the flask in a bowl of cold water.

2. Using a 50 ml measuring cylinder measure out 25 ml of conc. Sulphuric acid and carefully
add concentrated Sulphuric acid to the water. This mixture will heat up, so always place the
flask in a bowl of cold water while diluting the acid.

3. Mix gently and store it in amber colored bottle glass bottles with caps.

4. Label bottle with name of reagent (25 % Sulphuric acid), lot number, date of preparation,
expiration date & storage temperature.
5. Tighten caps & store at room temperature. Acid prepared can be stored upto 12 months
from the date of preparation

Preparation of 0.1% Methylene blue:

1. Using a digital balance weigh out 0.1 g of methylene blue in a sterile 500 ml flask and
dissolve it in 100 ml of distilled water.

2. Use a stirring bar or swirl solutions to mix.

3. Pour the solution through filter paper (Whitman No.1) and store filtered solution in an
amber colored glass bottles with caps.

4. Label bottle with name of reagent(0. 1 % Methylene blue), lot number, date of preparation,
expiration date & storage temperature.

5. Tighten caps & store at room temperature. Note: Store the reagent upto 6 months from
preparation date.
 ANNEXURE 2

PREPRATION OF LOWENSTEIN- JENSEN MEDIUM (LJ) MEDIA:

Principle:

Nitrogen and vitamin sources include L-Asparagine and Potato Flour. Monopotassium
Phosphate and Magnesium Sulphate are both growth promoters and buffers for organisms.
Malachite green inhibits the growth of most contaminants that survive specimen
decontamination while promoting the growth of Mycobacteria. Mycobacteria require fatty
acids and protein, which are provided by egg suspension. The egg albumin coagulates when
heated, creating a solid surface for inoculation. Glycerol is a carbon source that promotes the
growth of human tubercle bacillus while inhibiting the growth of bovine tubercle bacillus.

1. Dissolve 37.3 gm of the medium in 600 ml of distilled water containing 12 ml of

glycerol.
2. Heat if necessary to dissolve the medium completely.
3. Autoclave at 121°C for 15 minutes.
4. Prepare 1000 ml of a uniform suspension of fresh eggs under aseptic conditions.
Avoid whipping air into suspension during the collection and mixing.
5. Aseptically mix the 1000 ml of egg suspension with 600 ml of the sterile Lowenstein-
Jensen Medium cooled to 50 – 60°C, avoiding air bubbles.
6. Dispense the finished medium into sterile screw-cap test tubes.
7. Place the tubes in a slanted position and heat at 85°C for 45 minutes.
 ANNEXURE 3

ADENOSNE DEAMINASE ASSAY:

Adenosine deaminase (ADA) is a protein that is produced by cells throughout the body and
is associated with the activation of lymphocytes that play a role in the immune response to
infections. Conditions that trigger the immune system, such as an infection by
Mycobacterium tuberculosis, may lead to increase in the amount of ADA in the body fluids.
This test measures the amount of adenosine deaminase present in the body fluids in order to
help diagnose a tubercular infection of the area of body where the bacteria are present.
Detecting Mycobacteria in body fluids can be difficult because there may be large volume of
fluid and very low number of bacteria present. Though ADA is not a specific test and does
not replace the culture for diagnosing TB, it may be positive even when numbers of bacteria
are very low and can be used as an adjunct test to help determine whether tuberculosis is the
likely source of a person’s symptoms. The value of ADA is also increased in various
infectious diseases like infectious mononucleosis, typhoid, viral hepatitis, initial stages of
HIV and in case of malignant tumours. Reagents used: MICROXPRESS ADA-MTB is a
reagent for laboratory use only. ADA-TB comprises of:

a) L1- ADA-MTB Reagent- Buffer Reagent, ready to use.

b) L2- ADA-MTB Reagent- Adenosine Reagent, ready to use.

c) L3- ADA-MTB Reagent- Phenol Reagent.

d) L4- ADA-MTB Reagent- Hypochlorite Reagent.

e) S- ADA-MTB Standard- ADA Standard, ready to use.

Basic principle of the test is that Adenosine deaminase hydrolyses adenosine to ammonia
and inosine. The ammonia formed further reacts with a phenol and hypochlorite in an
alkaline medium to form a blue indophenol complex with nitroprusside acting as a catalyst.
Intensity of the blue coloured indophenol complex formed is directly proportional to the
amount of ADA present in the sample. Reagent preparation: Reagents L1, L2 and standard
are ready to use. Adenosine (L2) may form crystals at 2 C to 8 C. dissolve the same
gently warming (37 C - 50 C) the reagent for some time before use. Both the Phenol
reagent (L3) & Hypochlorite reagent (L4) need to be diluted 1:5 with distilled water before
use (1 part of reagent + 4 parts of distilled water). The working phenol reagent and working
hypochlorite reagent are stable for at least 6 months when stored at 2 C-8 C in tightly
closed bottles. ADA is reported to be stable in ascitic and PD Fluid samples for 2 days at 2
C-8 C, as after this, ammonia may be released in the samples even without any microbial
contamination.