Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2894_Библиотеки_им_академика_М_И_Перельмана
.pdf
60
https://t.me/medicina_free
R. Rai etal.
Table 5.1
Union/WHO scale
100× eld=HPF result
NEGATIVE 0 AFB/100 OIF 0 AFB/100 HPF 0 AFB/100 HPF
SCANTY 1–9 AFB/100 OIF 1–29 AFB/100 HPF 1–19 AFB/100 HPF
1+ 10–99 AFB/100 OIF 30–299 AFB/100 HPF 20–199 AFB/100
2+ 1–10 AFB/1 OIF (on
3+ >10 AFB/1 OIF (on
Source: NTEP Training Modules [58]
Grading scales for light (Ziehl-Neelsen) and uorescent microscopy
Bright eld (1000×
magnication)
average)
average)
Fluorescence (200–
250× magnication)
10–100 AFB/1 HPF 05–50 AFB/1 HPF
>100 AFB/1 HPF >50 AFB/1 HPF
Fluorescence (400×
magnication)
HPF
5.3.3 Reporting ofMicroscopy Results
The results of microscopy are analyzed using the grading scales as per WHO guidelines (Table5.1).
5.4 Advanced Microscopy forMycobacterial Research
While not currently recommended for TB diagnosis, there are several advanced
microscopy techniques, such as confocal laser scanning microscopy, uorescent
resonance energy transfer microscopy, single-molecule localization microscopy,
saturated structure illumination microscopy, time-lapse microscopy, and bioorthogonal corrective light electron microscopy, that are being used in TB research.
These techniques offer higher resolution, and some of them can be used to detect
Mtb in infected tissues. However, these methods require expensive equipment and
highly skilled professionals to operate them. In wide-eld microscopy, light/uorescence above and below the focal plane of the objective lens will also be detected
by the eyepiece or the detector. These signals from the out-of-focus regions add
blurs to the image, thus reducing the resolution. Laser scanning confocal microscopy (LSCM) provides a solution to this shortcoming. LSCM rejects any out-offocus light by focusing the illumination and detection optics on the same
diffraction-limited spot in the sample, such that only one spot of the sample is illuminated at a time. The confocal image of the sample is built up by sweeping a laser
beam across the sample via scanning galvanometer mirrors, and images are acquired
point by point. These images are then assembled together, and a complete image of
the sample/specimen is formed without any out-of-focus blurring. This technique
can generate both 2D and 3D images. LSCM excludes all nonspecic uorescence
by analyzing the emission spectra of each uorescence and omitting any uorescence other than those that do not emit a spectrum similar to the specic uorescent
markers used in the study. This conrms the specicity of the immunolabeling.
5 Different Methods ofMicroscopic andBacteriological Diagnosis ofTuberculosis
https://t.me/medicina_free
61
Erokhina etal. have demonstrated that LCMS-assisted immunohistochemical detection of Mtb using anti-TB polyclonal antibodies provides better resolution and specicity for Mtb detection in comparison to wide-eld immunouorescent
microscopy [59].
5.5 Conclusion
Bacteriologic and/or microscopic methods historically remained the mainstay of
TB diagnosis until very recently. Only after the advent of highly sensitive and specic molecular diagnostics tests that international governing bodies have started
discouraging the use of these methods because of the long turnaround time (culturebased diagnostics) and low sensitivity and specicity (culture/microscopy). It is
critical that results from these tests are further conrmed by using other biochemical/molecular methods, and repeat tests must be performed in patients who demonstrate clinical symptoms but tested negative by microscopy or culture. Despite all
these limitations, these classical methods remain relevant in resource-limited settings for the diagnosis of TB and are still the methods of choice for AST, surveillance, epidemiologic studies, and the assessment of treatment response. Efforts to
improve currently used media ordevelop newmedia that support faster growth of
Mtb and use of articial intelligence guided evaluation of microscopic slides are
innovations, which promise increase the utility of these methods in TB diagnosis
and help in the management of this global health threat.
References
1. Pigrau-Serrallach C, Rodríguez-Pardo D. Bone and joint tuberculosis. Eur Spine
J. 2013;22(4):556–66.
2. Choi EH, Coyle WJ.Gastrointestinal tuberculosis. Microbiol Spectr. 2016;4(6).
3. Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance [Internet]. [cited 2023 Mar 19]. https://www.who.int/
publications- detail- redirect/9789240028173.
4. Singh R, Dwivedi SP, Gaharwar US, Meena R, Rajamani P, Prasad T.Recent updates on drug
resistance in Mycobacterium tuberculosis. J Appl Microbiol. 2020;128(6):1547–67.
5. Eddabra R, Ait BH. Rapid molecular assays for detection of tuberculosis. Pneumonia.
2018;10:4.
6. MacLean E, Kohli M, Weber SF, Suresh A, Schumacher SG, Denkinger CM, etal. Advances in
molecular diagnosis of tuberculosis. J Clin Microbiol. 2020;58(10):e01582–19.
7. Schön T, Miotto P, Köser CU, Viveiros M, Böttger E, Cambau E.Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives. Clin Microbiol
Infect. 2017;23(3):154–60.
8. Diagnosis of tuberculosis disease [Internet]. [cited 2023 Mar 17]. https://www.cdc.gov/tb/edu-
cation/corecurr/pdf/chapter4.pdf.
9. Talbot EA, Adams LV, Fordham von Reyn C.The importance of culture for diagnosing tuberculosis. Clin Infect Dis. 2005;41(8):1213–4.
62
https://t.me/medicina_free
10. Canetti G, Froman S, Grosset J, Hauduroy P, Langerová M, Mahler HT, etal. Mycobacteria:
laboratory methods for testing drug sensitivity and resistance. Bull World Health Organ.
1963;29(5):565–78.
11. Training manual M tuberculosis C DST [Internet]. [cited 2023 Mar 19]. https://tbcindia.gov.
in/WriteReadData/l892s/6995271860Training%20manual%20M%20tuberculosis%20C%20
DST.pdf.
12. Kato-Maeda M, Metcalfe JZ, Flores L.Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies. Future Microbiol. 2011;6(2):203–16.
13. World Health Organization. Monitoring treatment response [Internet]. Companion handbook
to the WHO guidelines for the programmatic management of drug-resistant tuberculosis. 2014
[cited 2023 Mar 19]. https://www.ncbi.nlm.nih.gov/books/NBK247422/.
14. Kubica GP, Dye WE, Cohn ML, Middlebrook G. Sputum digestion and decontamination
with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am Rev Respir Dis.
1963;87:775–9.
15. Lowenstein Jensen medium [Internet]. [cited 2023 Mar 19]. https://legacy.bd.com/europe/
regulatory/Assets/IFU/Difco_BBL/244420.pdf.
16. Pfyffer GE, Wittwer F.Incubation time of mycobacterial cultures: how long is long enough to
issue a nal negative report to the clinician? J Clin Microbiol. 2012;50(12):4188–9.
17. Middlebrook G, Cohn ML.Bacteriology of tuberculosis: laboratory methods. Am J Public
Health Nations Health. 1958;48(7):844–53.
18. Middlebrook 7H10 Agar [Internet]. [cited 2023 Mar 18]. https://www.bd.com/resource.
aspx?IDX=9004.
19. Middlebrook 7H11 [Internet]. [cited 2023 Mar 18]. https://legacy.bd.com/europe/regulatory/
Assets/IFU/US/8801671(0703)en.pdf.
20. Middlebrooke 7H9 [Internet]. [cited 2023 Mar 17]. https://legacy.bd.com/europe/regulatory/
Assets/IFU/Difco_BBL/212352.pdf.
21. Lawson L, Emenyonu N, Abdurrahman ST, Lawson JO, Uzoewulu GN, Sogaolu OM, etal.
Comparison of Mycobacterium tuberculosis drug susceptibility using solid and liquid culture
in Nigeria. BMC Res Notes. 2013;6(1):215.
22. Park SK, Kim SC, Kim DM, Lee CW, Kim Y, Cho SN.Fully automated liquid culture system
compared with Lowenstein-Jensen solid medium for rapid recovery of Mycobacteria in sputums. Tuberc Respir Dis. 2002;53(6):635.
23. World Health Organization. Technical manual for drug susceptibility testing of medicines used
in the treatment of tuberculosis [Internet]. Geneva: World Health Organization; 2018 [cited 2023
Mar 19]. p.39. Report No.: 9789241514842. https://apps.who.int/iris/handle/10665/275469.
24. Heifets L, Linder T, Sanchez T, Spencer D, Brennan J.Two liquid medium systems, mycobacteria growth indicator tube and MB redox tube, for Mycobacterium tuberculosis isolation from
sputum specimens. J Clin Microbiol. 2000;38(3):1227–30.
25. Cauleld AJ, Wengenack NL.Diagnosis of active tuberculosis disease: from microscopy to
molecular techniques. J Clin Tuberc Mycobact Dis. 2016;4:33–43.
26. Tortoli E, Cichero P, Piersimoni C, Simonetti MT, Gesu G, Nista D.Use of BACTEC MGIT
960 for recovery of mycobacteria from clinical specimens: multicenter study. J Clin Microbiol.
1999;37(11):3578–82.
27. Pfyffer GE, Welscher HM, Kissling P, Cieslak C, Casal MJ, Gutierrez J, etal. Comparison of
the Mycobacteria Growth Indicator Tube (MGIT) with radiometric and solid culture for recovery of acid-fast bacilli. J Clin Microbiol. 1997;35(2):364–8.
28. Anargyros P, Astill DS, Lim IS. Comparison of improved BACTEC and LowensteinJensen media for culture of mycobacteria from clinical specimens. J Clin Microbiol.
1990;28(6):1288–91.
29. Chien HP, Yu MC, Wu MH, Lin TP, Luh KT.Comparison of the BACTEC MGIT 960 with
Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens. Int J
Tuberc Lung Dis. 2000;4(9):866–70.
R. Rai etal.
5 Different Methods ofMicroscopic andBacteriological Diagnosis ofTuberculosis
https://t.me/medicina_free
30. Diriba G, Kebede A, Yaregal Z, Getahun M, Tadesse M, Meaza A, et al. Performance of
Mycobacterium growth indicator tube BACTEC 960 with Lowenstein–Jensen method for
diagnosis of Mycobacterium tuberculosis at Ethiopian National Tuberculosis Reference
Laboratory, Addis Ababa, Ethiopia. BMC Res Notes. 2017;10(1):181.
31. Kumari P, Thakur JK, Kumar P, Kumar R, Parekh D.Comparison of LJ medium and BACTEC
MGIT 960 culture system for the diagnosis of tuberculosis. J Clin Diagn Res [Internet]. 2020
[cited 2023 Mar 17]. https://jcdr.net/article_fulltext.asp?issn=0973- 709x&year=2020&volum
e=14&issue=12&page=DC09&issn=0973- 709x&id=14304.
32. Palomino JC.Nonconventional and new methods in the diagnosis of tuberculosis: feasibility
and applicability in the eld. Eur Respir J. 2005;26(2):339–50.
33. Gravet A, Souillard N, Habermacher J, Moser A, Lohmann C, Schmitt F, etal. Culture and
susceptibility testing of mycobacteria with VersaTREK.Pathol Biol (Paris). 2011;59(1):32–8.
34. Espasa M, Salvadó M, Vicente E, Tudó G, Alcaide F, Coll P, etal. Evaluation of the VersaTREK
system compared to the Bactec MGIT 960 system for rst-line drug susceptibility testing of
Mycobacterium tuberculosis. J Clin Microbiol. 2012;50(2):488–91.
35. Yuksel P.Comparison of the VersaTrek and BACTEC MGIT 960 systems for the contamination rate, time of detection and recovery of mycobacteria from clinical specimens. Afr J
Microbiol Res [Internet]. 2011 [cited 2023 Mar 17];5(9). http://www.academicjournals.org/
ajmr/abstracts/abstracts/abstract%202011/4May/Yuksel%20et%20al.htm.
36. Falconi FQ, Infante Suárez L, López MJ, García SC.Comparison of the VersaTREK system
and Löwenstein-Jensen medium for the recovery of mycobacteria from clinical specimens.
Scand J Infect Dis. 2008;40(1):49–53.
37. Piersimoni C, Scarparo C, Callegaro A, Tosi CP, Nista D, Bornigia S, etal. Comparison of MB/
BacT ALERT 3D system with radiometric BACTEC system and Löwenstein-Jensen medium
for recovery and identication of mycobacteria from clinical specimens: a multicenter study. J
Clin Microbiol. 2001;39(2):651–7.
38. Ängeby KAK, Werngren J, Toro JC, Hedström G, Petrini B, Hoffner SE.Evaluation of the
BacT/ALERT 3D system for recovery and drug susceptibility testing of Mycobacterium tuberculosis. Clin Microbiol Infect. 2003;9(11):1148–52.
39. Martinez MR, Sardiñas M, Garcia G, Mederos LM, Díaz R.Evaluation of BacT/ALERT 3D
system for mycobacteria isolates. J Tuberc Res. 2014;02(02):59–64.
40. WHO consolidated guidelines on tuberculosis: module 3: diagnosis: rapid diagnostics for
tuberculosis detection, 2021 update [Internet]. [cited 2023 Mar 17]. https://www.who.int/
publications- detail- redirect/9789240029415.
41. Desikan P. Sputum smear microscopy in tuberculosis: is it still relevant? Indian J Med Res.
2013;137(3):442–4.
42. Maynard-Smith L, Larke N, Peters JA, Lawn SD.Diagnostic accuracy of the Xpert MTB/RIF
assay for extrapulmonary and pulmonary tuberculosis when testing non-respiratory samples: a
systematic review. BMC Infect Dis. 2014;14(1):709.
43. Hepple P, Ford N, McNerney R.Microscopy compared to culture for the diagnosis of tuberculosis in induced sputum samples: a systematic review [review article]. Int J Tuberc Lung Dis.
2012;16(5):579–88.
44. Chawla K, Gupta S, Mukhopadhyay C, Rao PS, Bhat SS.PCR for M. tuberculosis in tissue
samples. J Infect Dev Ctries. 2009;3(2):83–7.
45. Rufai SB, Kumar P, Singh A, Prajapati S, Balooni V, Singh S.Comparison of Xpert MTB/RIF
with line probe assay for detection of rifampin-monoresistant Mycobacterium tuberculosis. J
Clin Microbiol. 2014;52(6):1846–52.
46. Raizada N, Sachdeva KS, Nair SA, Kulsange S, Gupta RS, Thakur R, etal. Enhancing TB case
detection: experience in offering upfront Xpert MTB/RIF testing to pediatric presumptive TB
and DR TB cases for early rapid diagnosis of drug sensitive and drug resistant TB.PLoS One.
2014;9(8):e105346.
47. Tuberculosis: standard treatment guidelines [Internet]. [cited 2023 Mar 18]. https://www.nhm.
gov.in/images/pdf/guidelines/nrhm- guidelines/stg/stg- tb.pdf.
63
64
https://t.me/medicina_free
48. Singhal R, Myneedu VP. Microscopy as a diagnostic tool in pulmonary tuberculosis. Int J
Mycobacteriol. 2015;4(1):1–6.
49. Allen JL.A modied Ziehl-Neelsen stain for mycobacteria. Med Lab Sci. 1992;49(2):99–102.
50. RNTCP Lab Network Guidelines.pdf [Internet]. [cited 2023 Mar 18]. https://tbcindia.gov.in/
WriteReadData/l892s/4234099618RNTCP%20Lab%20Network%20Guidelines.pdf.
51. Lahiri K, Chatterjee S. A simple cold staining method for acid fast bacilli. Med J Armed
Forces India. 1994;50(4):256–8.
52. Riley PA. Principles of microscopy, culture and serology-based diagnostics. Medicine
(Baltimore). 2017;45(10):639–44.
53. World Health Organization. Fluorescent light-emitting diode (LED) microscopy for diagnosis
of tuberculosis: policy statement [Internet]. World Health Organization; 2011 [cited 2023 Mar
18]. Report No.: WHO/HTM/TB/2011.8. https://apps.who.int/iris/handle/10665/44602.
54. Lichtman JW, Conchello JA.Fluorescence microscopy. Nat Methods. 2005;2(12):910–9.
55. Young MR. Principles and technique of uorescence microscopy. J Cell Sci.
1961;s3-102(60):419–49.
56. Reza LW, Satyanarayna S, Enarson DA, Kumar AMV, Sagili K, Kumar S, et al. LEDuorescence microscopy for diagnosis of pulmonary tuberculosis under programmatic conditions in India. PLoS One. 2013;8(10):e75566.
57. Flourescence_Microscopy Manual.pdf [Internet]. [cited 2023 Mar 18]. https://tbcindia.gov.in/
WriteReadData/l892s/7890638455Flourescence_Microscopy%20Manual.pdf.
58. NTEP training modules [Internet]. [cited 2023 Mar 19]. https://tbcindia.gov.in/WriteReadData/
NTEPTrainingModules1to4.pdf.
59. Erokhina MV, Nezlin LP, Avdienko VG, Voronezhska EE, Lepekha LN.Immunohistochemical
detection of Mycobacterium tuberculosis in tissues of consumptives using laser scanning
microscopy. Biol Bull. 2016;43(1):21–5.
60. WHO. Tuberculosis (TB) [Internet]. 2022 [cited 2023 Mar 17]. https://www.who.int/
news- room/fact- sheets/detail/tuberculosis.
R. Rai etal.
Chapter 6
https://t.me/medicina_free
Molecular Diagnosis ofTuberculosis
SanjaySinghNegi, PushpendraSingh, andKuldeepSharma
Abstract Tuberculosis (TB) is a global health problem as it causes a signicantly
large number of new cases and mortality every year across the world. Its magnitude
is well understood for its clearity of enhanced deteriorating effect on public health.
Accordingly, authors is requesting for retaining the sentence as such due to various
factors including the emergence of drug resistance cases (multidrug, extensive and
total drug resistance TB), overcrowding and poverty forcing poor nutrition and various precipitating infectious agents like HIV, HCV, leishmania enabling
Mycobacterium tuberculosis (MTB) complex to sets in infection in patients coinfected with these agents. Thus, early, accurate, and specic diagnosis is required for
the initiation of specic antitubercular treatment (ATT) for effective management.
Although conventional clinical, microscopic, and radiological diagnostic approaches
are indispensable, they have their limitations. Further, the low sensitivity of conventional diagnostic approach based on microscopy and culture and non-recommended
serological tests has warranted the utmost need to develop more rapid and accurate
tests for the diagnosis of MTB complex. The slow generation time of 16–18 hours
of MTB restrict the utility of culture for diagnosis as it takes an average of 3-4
weeks to maximum of 6-8 weeks for conrmation. Conventional polymerase chain
reaction (PCR), real-time PCR (RT-PCR), loop-mediated isothermal amplication
(LAMP) assay, line probe assay (LPA), automated systems like GeneXpert and
TruNAT, and whole genome or target sequencing have made a tremendous advancement in the diagnosis of both pulmonary and extrapulmonary TB. The unique
advantage has already been witnessed and reported in extrapulmonary TB, wherein
the paucibacillary nature of the bacilli has made conventional approach almost
impossible to diagnose it. This book chapter accordingly highlights the recent
development witnessed in the molecular diagnosis of both TB and drug resistance.
Keywords Tuberculosis · PCR · Xpert · TruNAAT · LAMP · Drug resistance
S. S. Negi (*) · P. Singh · K. Sharma
Department of Microbiology, All India Institute of Medical Sciences (AIIMS),
Raipur, Chhattisgarh, India
e-mail: negidr@aiimsraipur.edu.in
© 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_6
65
66
https://t.me/medicina_free
S. S. Negi etal.
6.1 Introduction
Tuberculosis (TB) is still being recognized as the leading infectious disease globally. The recent global TB report, 2022, has reported 1.6 million deaths and 10.6
million new cases worldwide due to TB in 2021 [1]. The situation is further aggravated by an increase in the number of cases of drug-resistant TB.The World Health
Organization (WHO) has reported 450, 000 new cases of rifampicin (RIF)-resistant
TB in 2021 [1]. Furthermore, isoniazid (INH), another important drug for treatment,
was reported to have a resistance of 7.1% in new cases, along with 7.9% in previously treated TB cases [1].
TB is caused by Mycobacterium tuberculosis (M. tuberculosis) complex (MTB),
which comprises M. tuberculosis (human tubercle bacillus, MTB), M. bovis (bovine
tubercle bacillus), M. canetti (reported from a few cases in East Africa), M. africa-
num (reported in a few cases from West Africa), M. caprae (closely related to
M. bovis), M. microti (vole bacillus, rare and less virulent), and M. pinnipedii
(recently isolated from humans). Among all of them, MTB is the most common
causative agent of TB.The most common manifestation of MTB is pulmonary TB
(PTB), wherein the lung is the affected site. However, through a hematogenous
route, it can disseminate to other organs to cause extrapulmonary TB (EPTB).
Virtually, except hair, it can affect any organ. The commonest site is the lymph
nodes (posterior cervical and supraclavicular lymph nodes), to cause tubercular
lymphadenitis, pleura(pleural TB), upper airways (laryngx, pharynx, and epiglottis), genitourinary TB (renal TB and genital TB in which the fallopian tube and
endometrium are commonly involved in females to cause infertility, while in males,
the epididymis is the most affected site), skeletal TB, tubercular meningitis, gastrointestinal TB, tubercular pericarditis, skin tubercular lesin of scrofuloderma and
lupus vulgaris, and military or disseminated TB.The management of TB is an uphill
task since it requires multi-antimicrobial therapy for a longer period of time, ranging from a minimum of 6months up to even 2years. The management is severely
hampered by the emergence of various drug-resistant forms of bacilli, like multidrugresistant TB (MDR-TB), extensive drug-resistant TB (XDR-TB), and total drugresistant TB (TDR-TB). Accordingly, better and appropriate management of TB
utmost requires an early, sensitive, and specic diagnosis of MTB to initiate optimal
treatment in positive cases to prevent its further spread and to reduce associated
mortality and morbidity [2].
6.2 Laboratory Diagnosis
The diagnosis of TB includes conventional, radiographical, and molecular diagnostic modalities. Since this chapter focuses on the molecular diagnosis of TB, the
conventional approach is very briey discussed.
6 Molecular Diagnosis ofTuberculosis
https://t.me/medicina_free
67
6.3 Conventional Methods fortheDiagnosis ofTB
Conventional diagnosis is the most widely used microscopy post Ziehl-Neelson
(ZN) or uorescent staining and culture for the isolation of MTB on either solid
culture medium like Lowenstein Jensen (LJ Media) or liquid culture system like
Mycobacteria Growth Indicator Tube (MGIT-Automated). Microscopy is a rapid
test and may be completed within half an hour to detect acid-fast bacilli (AFB) in
the smear examination. Since its limit of detection requires a minimum bacterial
load of 10,000/mL of clinical sample, it lacks sensitivity and specicity [2]. It further does not differentiate between various Mycobacterium species [2]. Culture, on
the other hand, is considered a gold standard as it provides specic growth identication features to identify MTB.However, although it is more sensitive than any
other conventional methods, it is time-consuming. Culture using LJ media requires
4–8weeks while the liquid culture system around 2–8weeks. Further, it requires
laboratory infrastructure and trained staff. The identication of antitubercular (ATT)
drug sensitivity patterns requires another 4 to 6weeks. Therefore, the utmost need
for a new rapid, sensitive, and specic test was felt. The World Health Organization’s
(WHO’s) End TB Strategy has set a goal of 95% reduction in TB death and 90%
reduction in incidence rate by 2035. This ambitious plan for TB elimination highlights the critical role of laboratories in providing an early, sensitive, and specic
diagnosis of TB and universal drug susceptibility testing to determine the MTB
sensitivity pattern against antitubercular drugs. Various drug-resistant forms of TB,
like monodrug resistant-, multidrug-resistant (MDR)-, XDR-, and TDR-TB, have
further warranted the consistent demand for an upgrade in conventional drugsusceptibility testing (DST) to provide fast and reliable sensitivity patterns of the
MTB [3].
6.4 Molecular Methods fortheDiagnosis ofTB
Molecular diagnosis, in the last one/two decades, has provided a revolutionary
improvement in the eld of both the detection and sensitivity testing of
MTB.Presently, a variety of nucleic acid amplication test (NAAT)-based assays
are being used to determine the drug susceptibility of MTB for the effective treatment of TB patients. DST-MTB, in relation to the “critical concentration,” compares
the growth of susceptible versus resistant strains [4]. The discrepancies among genotype and phenotype DST approaches, which are limited mainly to certain antibiotics, need to be resolved by more research into resistance mechanisms [5]. The
phenotypic and genotypic methods should be used to dene the antibiotic resistance
prole to treat patients with MDR-TB and XDR-TB in the era of drug resistance
MTB [6].
Rifampicin resistance is a suboptimal surrogate for MDR-TB. Resistance to
rifampicin (RIF) of more than 95% in MTB is principally connected to changes
within 81bp (codons 507 to 533) RIF resistance-determining region (RRDR) in the
68
https://t.me/medicina_free
S. S. Negi etal.
rpoB gene [7, 8]. Earlier studies have shown that mutations in the rpoB RRDR are
more probable to deliberate high levels of RIF resistance [8–10]. Single nucleotide
changes are the domination mutations, resulting in mutations of particular amino
acid residues at positions 516, 526, and 531. In-frame insertion and deletion changes
also happen, although at lesser incidences at residue positions 511, 516, 518, and
522. In the rpoB gene, especially the RRDR region, one deletion, two insertions,
ve multiple mutations (with two or three concerned codons), and 16 single base
mutations, were reported [11, 12](Table 6.1).
Resistance to isoniazid (INH) is a complex process that generates mutations/
substitutions/deletions in various genes, like katG, inhA, ahpC, kasA, and ndh,
which have all been related to INH resistance [13]. The INH is a drug needing activation by the catalase/peroxidase protein that encode by katG. This activation of
INH is severly hampered due to the frequently reported mutation S315T in the katG
gene resulting in drug resistance against INH.Another mechanism contains mutations in the inhA promoter at C15T, which results in an excess expression of inhA,
which shows low-level INH resistance [14]. Torres etal. exposed new changes in
sequence that are capable of elucidating 98% of phenotypic INH resistance through
katG, inhA promoter substitution [15](Table 6.1).
Resistance to pyrazinamide (PZA) is primarily caused by mutations in the pncA
gene of MTB.The mutations are remarkably situated and spread throughout the
pncA gene, which encodes the pyrazinamidase protein or enzyme, which activates
the drug [16]. Different studies on PZA resistance had reported the clustered mutations in the three conserved regions at amino acids positions 3–71, 61–85, and
132–142 most responsible for drug resistance against PZA. [17–19](Table 6.1).
Groups of PZA-resistant MTB with identical pncA mutations have been reported
but so far have been geographically restricted [19]. Studies have reported PZA resistance of more than 90% in XDR-TB isolates and about 40–50% in primary
MDR-TB [20].
Ethambutol (EMB) is a bacteriostatic agent that acts against MTB by preventing
membrane-related arabinosyl transferases, which are encoded by the embCAB
operon (embA, embB, and embC) [21]. Earlier studies exhibited that common EMBresistant clinical isolates that have mutations within embB, mainly at position 306,
could be used as resistance markers for the fast detection of EMB resistance [22].
Some of the changes within the upstream region of embA are similarly connected
with EMB resistance [21] (Table6.1).
Molecular techniques have seen a revolutionary evolution in the last two decades
for the diagnosis of MTB and the determination of drug resistance prole with the
emergence of various molecular tests, like polymerase chain reaction (PCR), realtime PCR, loop-mediated isothermal amplication (LAMP), TruNat, GeneXpert,
specic target nucleotide sequencing, and next-generation sequencing (NGS).
This chapter has thus attempted to summarize the existing molecular diagnostic
tests and the future aspects in the eld of detection of MTB and DR-MTB.Instead
of exhaustive detail of all the commercial NAAT tests, the chapter provides the
basic priniciples of the tests along with their sensitivity, specicity, TAT, advantages
and limitations.
6 Molecular Diagnosis ofTuberculosis
https://t.me/medicina_free
Table 6.1 Anti-mycobacterial drugs and mechanisms of drug resistance [23]
Most
S.
no. Agent Mode of action Target Gene
1 Isoniazid Inhibition of
mycolic acid
synthesis
Drug target
alteration
Drug target
overexpression
2 Rifampicin Inhibition of
RNA polymerase
3 Pyrazinamide Inhibition of
energy production
and
trans-translation
4 Ethambutol Inhibition of
arabinogalactan
synthesis
5 Streptomycin Binds to the small
16S rRNA of the
30S ribosomal
subunit
irreversibly, along
with protein S12
6 Fluoroquinolone Inhibition in
cleaved
complexes
7 Ethionamide Inhibition in fatty
acid biosynthesis
pathway
Inhibits the cell
wall synthesis
Mycolic acids katG Ser-315-
Enoyl-ACP
reductase
Enoyl-ACP
reductase
promoter
RNA polymerase rpoB Ser-450-
Fatty acid
synthase-I,
ribosomal
protein S1
Arabinosyl
transferases
Ribosomal
protein S12
DNA gyrase gyrA Ala-90-
Enoyl-ACP
reductase
NADPH-specic
FAD-containing
monooxygenase
inhA Asn-146,
inhA
promotor
pncA Asp-12-
embCAB Met-306-
rpsL Lys-43-
inhA Asn-146,
EthA Val202-
prevalent
mutation
Thr
Asn-268,
and
Asn-302
Cys-15-
Thr
Leu
Ala/Asn,
Leu-85-
Pro
Val/Ile/
Leu
Arg
Val,
Ser-91-
Pro, and
Asp-94-
(Gly/Ala/
His/Asn)
Asn-268,
and
Asn-302
Leu
69
Frequency
in resistant
strain
60–70
>95
70–100
69
60
>90
<10
Соседние файлы в папке Библиотека им академика М.И. Перельмана