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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2894_Библиотеки_им_академика_М_И_Перельмана
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Abbreviations
TNF Tumor necrosis factor
TNF-α Tumor necrosis factor-alpha
TST Tuberculin skin test
TTD Time to detection
TU Tuberculin unit
UCNPs Upconversion Nanoparticles
U-DST Upfront Universal-Drug Sensitivity Testing strategy
UNION International Union Against Tuberculosis and Lung Disease
USG Ultrasonography
UV Ultraviolet
VNTR Variable number of tandem repeats
VOBa Volatile organic biomarkers
WBCs White blood cells
WGS Whole genome sequencing
WHO World Health Organization
XDR Extensively drug resistance
ZN Ziehl-Neelsen
zTB Zoonotic tuberculosis
Chapter 1
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Introduction totheDiagnosis
ofMycobacterium
AmitSingh, GuruDuttaSatyarthee, andDivakarSharma
Abstract Mycobacterium tuberculosis (Mtb) is a causative agent of tuberculosis. It
is slightly curved, rod shaped, and arranged in singles or in groups. It manifests as
pulmonary, extrapulmonary, and sometimes miliary disease. It grows very slowly
and takes up to 21–42days to become a visible colony in the Löwenstein-Jensen
(LJ) growth media. Cases of tuberculosis are increasing, and timely management is
an essential component for the control of the disease, in which laboratory diagnosis
plays an important role. Various diagnostics tools have been developed and used for
the diagnosis of tuberculosis, drug-resistant tuberculosis, nontuberculosis mycobacterium (NTM), and zoonotic tuberculosis. The available techniques involved
more ancient like microscopy, culture to the mostadvanced molecular tests, matrixassisted laser desorption/ionization-time of ight-mass spectrometry (MALDITOF- MS), and point-of-care (POC) devices (e.g., LAM). These available tools are
currently used in the management of tuberculosis. Researchers are also exploring
newer, advanced, fast, and cheaper diagnostics for tuberculosis to overcome the current limitations. In the introductory chapter, we outline and summarize the different
laboratory tests used for the diagnosis of tuberculosis, which are further elaborated
in the dedicated chapter of the book.
Keywords Tuberculosis · LJ culture · Microscopy · GeneXpert · NAAT ·
Automated culture · Zoonotic tuberculosis · NTM
A. Singh (*)
Department of Gastroenterology & HNU, All India Institute of Medical Sciences,
New Delhi, India
Department of Microbiology, Central University of Punjab, Bathinda, India
G. D. Satyarthee
Department of Neurosurgery, All India Institute of Medical Sciences, New Delhi, India
D. Sharma
Department of Microbiology, Maulana Azad Medical College, New Delhi, India
© The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2023
A. Singh, D. Sharma (eds.), Diagnosis of Mycobacterium,
https://doi.org/10.1007/978-981-99-5624-1_1
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A. Singh etal.
1.1 Introduction
In 2022, tuberculosis (TB) caused an estimated 1.6 million deaths, including
187,000 deaths among people positive with HIV/AIDS and 1.4 million among HIV
negatives, and gave rise to 10.6 million new cases globally, becoming the leading
infectious cause of mortality in the world [1]. The number of new cases of rifampicin resistant was reported to be 450,000in 2021. Approximately three to ve million cases are not reported or diagnosed, which might have contributed to the
increase in tuberculosis cases globally [1]. The most common manifestation of
tuberculosis is known as pulmonary tuberculosis (PTB), which mainly infects the
lungs. The bacterium may spread to other organs through a hematogenous route and
may cause infection in other organs; this is called extrapulmonary tuberculosis
(EPTB) [2]. Depending on the site of infection, various clinical samples were collected. In PTB cases, sputum, induced sputum, bronchoalveolar lavage (BAL), etc.
were collected, and for EPTB cases, pleural uid, pericardial uid, blood, bone
marrow, cerebrospinal uid (CSF), tissue, pus, swabs, urine, endometrium aspirate,
lymph nodes, ne-needle aspiration cytology (FNAC) samples, bronchial secretions, and gastric lavage [3, 4]. Depending on the samples, different diagnostic tech-
niques were used to diagnose tuberculosis.
The diagnosis of TB includes conventional (culture, Ziehl-Neelsen (ZN), and
uorescent stained microscopy) [5], radiological (X-ray, CT scan, MRI) [6], and
molecular diagnostics (GeneXpert, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), line probe assay (LPA)) [7–11].
Microscopy is a rapid test and may be completed within half an hour to detect acidfast bacilli (AFB) in smear examination [12]. Since its limit of detection requires a
minimum bacterial load of 10,000/ml of clinical sample, it lacks sensitivity and
specicity [10, 13, 14]. Further, it does not differentiate between infectious and
environmental mycobacteria. The culture method is considered a gold standard for
tuberculosis diagnosis as it provides a specic growth identication feature to characterize up to species level and can further be used for drug susceptibility tests [15,
16]. The culture technique is more sensitive and more specic than other conven-
tional diagnostics methods but is time-consuming. Mycobacterium tuberculosis
grows very slowly, and Löwenstein-Jensen (LJ) culture takes 4–8weeks, whereas
automated liquid culture methods take 2–6weeks [17]. The molecular test is rapid,
sensitive but requires laboratory infrastructure, is expensive, and needs trained manpower to perform it [13]. Further research needs to identify novel biomarkers for the
development of rapid, inexpensive, and sensitive point-of-care tests for the effective
management of tuberculosis and to achieve World Health Organization’s (WHO’s)
sustainable development goal (SDG) to end TB [1, 18]. The major challenges for
thediagnosis of zoonotic tuberculosis [19], TB in pediatric cases and vulnerable
populations [9, 20].
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1.2 Laboratory Diagnosis ofMycobacterium
Laboratory diagnosis starts with sample collection. The identication of the appropriate site of infection and collecting samples for the diagnosis of mycobacterium
are important tasks. The procedure and quality of the sample determine the correct
laboratory diagnosis of the disease. The most commonly used sample for Mtb culture is sputum from patients with pulmonary TB [4, 17]. For EPTB, various clinical
samples were collected depending on the site of infection on sterile container [3,
17]. The samples include tissue, pleural uid, pericardial uid, blood, bone marrow,
CSF, tissue, pus, swabs, urine, endometrium aspirate, lymph nodes, FNAC samples,
bronchial secretions, and gastric lavage. After receiving the samples, proper storage
conditions and procedures should be followed to proceed to diagnostics. An appropriately collected sample is the key to the diagnosis of TB [17]. The collected samples were further processed for mycobacterial diagnosis, depending on the facility
available. The diagnosis of tuberculosis is divided into conventional, radiological,
and molecular methods; immunological diagnosis; and newer diagnosis (Figs.1.1
and 1.2).
1.3 Conventional Methods
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1.3.1 Microscopy
Smear microscopy is the most rapid and a cost-effective method for the detection of
tuberculous bacilli [17]. The smears are prepared from processed or direct samples
and further stained with ZN stain or uorescent stained and observed under standard
light microscopy and a uorescent microscope, depending on the staining techniques used. The ZN-stained tubercle bacilli under a light microscope appear as red
pink, long, or slightly curved bacilli [17]. The rhodamine-auramine stained bacilli,
when observed under a uorescent microscope, appears yellow or orange under
ultraviolet light and bright orange bacilli in pale green background when stained by
acridine orange [17]. Some histopathological methods (e.g., immunohistochemistry
(IHC)) were also used for the diagnosis of tuberculosis.
1.3.2 Culture
The culture method is more sensitive and to date is considered a gold standard
method for the diagnosis of tuberculosis. Culture is also essential for performing
drug-susceptibility testing (DST) and genotyping through mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), restriction
fragment length polymorphism (RFLP), or spoligotyping methods [12, 21, 22].
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Molecular Te st
RT-PCR based
PCR based diagnosis
Line Probe Assay
GeneXpert
TruNAAT
LAMP test
A. Singh etal.
Culture based
LJ Culture
MGIT Culture
BactAlertCulture system
Fast Plaque assay
Microscopy
AFB staining
AR staining
Laboratory Diagnosis
of tuberculosis
Point-of-Care
LAM-IC T
Mpt64 for NTM
GeneXpert
Newer Test
MALDI- TOF-MS
VOC-test
IC T based
Immunological
Mantoux test
IGRA test
Fig. 1.1 Laboratory diagnosis of mycobacterium
Sterile samples were directly inoculated on culture media, while sputum or nonsterile samples were processed through Patroff’s modied methods and then incubated
at 37°C for up to 8weeks [17]. Mtb shows rough, tuff, buff, and creamy colonies in
the LJ medium [5, 17]. In the liquid culture medium, the bacilli grow on surface and
form cordsand wrinkle pellicle. They grow more rapidly in liquid culture media as
compared to LJ or other solid media. Automated culture systems (Mycobacteria
Growth Indicator Tube (MGIT), BactAlert, VersaTREK, etc.) are more rapid, and
growth is detected through the production of uorescence or a change in color due
to the depletion of carbon dioxide (CO2) in the medium [15, 17, 23]. Further Mtb
identication in culture is performed through biochemical tests (nitrate reductase,
niacin test, arylsulfatase, urease, and tween 80 hydrolysis), molecular tests, or
immunochromatographic test (ICT)-based MPT64 strip-based tests (Fig.1.1) [17].
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Fig. 1.2 Flow chart of various dianostics methods for the diagnosis of mycobacterium
The bacteriophage-based test was also developed to detect TB in sputum samples
[17]. Nonpathogenic mycobacteria (i.e., sensor cells) were used as control bacteria
in the test. The phage replicates, infects, and lyses the sensor cells, leaving zones of
clearing (holes) in the agar media. The zone of clearing indicates that the patient’s
sputum contains viable TB bacilli. The test is cheaper, sensitive (only requires 100
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bacilli/ml sputum), and fast with a 2-day turnaround time. Its main limitations are it
is technically demanding and can be applied for sputum specimens only.
A. Singh etal.
1.4 Radiological Methods
Radiological diagnosis is an important tool for the diagnosis of PTB and
EPTB. Clinicians must learn this skill and sharpen it to improve TB diagnosis.
Errors in radiograph interpretation can lead to both under- and overdiagnosis.
Remember impact of rotation, phase of respiration, exposure, motion, artifact and
cofounders like thymic shadow. Radiographic ndings in tuberculosis have been
well described [24]. Chest X-ray (CXR), CT scan, and MRI are most commonly the
radiological methods used for the presumptive diagnosis of tuberculosis (Fig.1.2).
1.5 Molecular Diagnosis
Earlier methods are either less sensitive, time-consuming, and have a slow growth
rate of Mtb, whereas molecular methods have become the rst choice for the rapid
detection of TB.Various Nucleic Acid Amplication Test (NAAT)-based tests were
developed for the detection of tuberculosis directly from patient samples. These
tests can identify etiological agents and also provide information on drug resistance.
The cartridge-based GeneXpert and TruNAAT are the current choice for molecular
diagnosis because of their ease of performance, but these are very expensive and
need infrastructure. PCR or multiplex polymerase chain reaction (mPCR) is the
most common format of NAAT; other amplication techniques include loopmediated isothermal amplication (LAMP), ligase chain reaction (LCR), strain displacement amplication, CRISPER-Cas, and real-time-based tests. Some molecular
tests, like Xpert MTB/RIF, Xpert MTB/XDR, LPA, and real-time-based tests are
capable of providing information on drug resistance. Whole-genome sequencing or
targeted sequencing methods are also in use for the identication and detection of
TB either from samples or culture [10, 11, 23, 25, 26].
1.6 Immunological Methods
Immunological methods used for the immunodiagnostics of TB can provide indirect
evidence on the current or past infections/exposure to tubercle bacilli. Tuberculin
skin test (TST) has limited application due to cross-reactivity and poor sensitivity
[8, 13]. Also, WHO bans all existing serological tests for use in the diagnosis of
tuberculosis due to their poor sensitivity and specicity and at the same time
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encourages identifying novel biomarkers to develop newer immunodiagnostics for
the detection of tuberculosis [14, 27].
1.6.1 Antigen Detection
The detection of tuberculosis antigens in patient samples provides direct evidence
of TB.Lipoarabinomannan (LAM) is a promising antigen for antigen detection for
TB and is used as a point-of-care test for the rapid detection of TB in urine and other
samples. LAM is a promising target for antigen detection for TB diagnosis due to
its temperature stability and its possibility to be detected in urine [28]. LAM-based
assays are included in the WHO-TB diagnosis retooling program.
1.6.2 Antibody Detection
Antibody detection against TB is rapid, cost-effective, simple, and easy to perform,
but due to its poor sensitivity and specicity, WHO bans the use of serological tests
for tuberculosis diagnosis and instructed researchers to nd novel biomarkers for
the development of antibody-based detection methods [13].
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1.6.3 Tuberculin Skin Test or PPD Test
The PPD (puried protein derivative) test determines previous exposure to TB
bacilli or whether the patient received bacillus Calmette-Guérin (BCG) vaccination.
The standard recommended Mantoux test, in which 0.1mL of liquid containing 5
TU (tuberculin units) PPD is injected into the top layers of skin of the forearm and
observed for presence or absence and the amount of induration (localized swelling)
within 48–72h after the injection [9].
1.6.4 Interferon-Gamma Release Assays (IGRAs)
IGRA overcomes the problems confronting tuberculin skin test (TST) interpretation. There are two available formats for IGRA: (1) Quantiferon-TB Gold and (2) T
Spot-TB test. IGRA assay is based on the ability of TB antigens, such as ESAT-6
and CFP-10, to stimulate the host for the production of interferon-gamma (IFN-γ).
The amount of interferon-γ released from the cells is estimated. Results are interpreted both qualitatively (positive, negative, or indeterminate) and quantitatively.
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Immunization with BCG does not affect the test result of the IGRA assay because
the antigens used are specic to Mtb and absent in BCG [29].
A. Singh etal.
1.7 Newer TB Diagnosis
Rapid technological development in the laboratory diagnosis of tuberculosis, especially molecular diagnosis, has reduced the time required for identication and susceptibility testing. Continuous effort is made for the increase of reproducibility,
improvement of performance, and cost-effectiveness. Many rapid, portable, and
sensitive biosensors, electro-sensors with instant “on-the-spot” interpretation, have
been developed for tuberculosis detection based on different biological elements,
like DNA, RNA, or protein recognition systems and basic signal transducer principles. The combination of biosensing technology and nanotechnology is very promising. Many point-of-care devices are in development that will further help in
enhancing TB detection [13].
Currently, nanoparticles have shown a potential for the diagnosis of infectious
diseases. The engineering of biomaterials and nanocarriers represents an exciting
opportunity in the eld of diagnostics (30). Various innovative and efcient nanodiagnostics have been developed by researchers for infectious diseases, including
TB.Laksanasopin etal. [31] developed a smartphone-based POC to diagnose infectious diseases by connecting traditional immunoassay into a smartphone via accessories such as dongles [13, 31, 32].
1.8 Conclusions
This is now exciting era for development of newer TB diagnostics. The development of various advanced biosensors and nanotechnology enhance the possibility to
developing rapid, sensitive point-of-care tests. Till now, rapid, high-specic molecular assays for TB detection and drug susceptibility tests cannot replace the standard
diagnostic methods (such as culture, microscopy, clinical and radiological assessments, and conventional DST for active TB in PTB and EPTB samples). In this
book, we are covering all the diagnostics of Mycobacterium, from microscopy to
the more advanced point-of-care tests for the detection of tuberculosis.
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