18

Mycobacterium
MYCOBACTERIUM TUBERCULOSIS Secondary TB
yy Occurs in adults (latent infection – reactivation)
•• Acid fast organism yy It mainly affects the upper lobe
•• Gram-positive bacilli yy It leads to necrosis, tissue destruction and cavity formation
•• Slender rods yy They expectorate plenty of bacilli leading to spread of infec-
•• Has characteristic presence of mycolic acid in cell wall tion
•• Slow growing, nonmotile, and noncapsulated yy Widespread dissemination occurs in immunodeficient indi-
viduals
yy Mycobacterium tuberculosis differs from other organisms by
not being visualized in Gram staining
yy It needs special staining called as acid-fast staining
yy Discovered by Robert Koch
yy Acid fastness is because of presence of unsaponifiable lipid-
rich waxy mycolic acid that is seen in the cell wall
yy M. tuberculosis is both acid (20–25% sulphuric acid) and
alcohol (3% hydrochloric acid) fast

Remember
Acid fast organisms:
•• Mycobacterium
•• Nocardia
•• Rhodococcus
•• Legionella micdadei
•• Coccidiean parasites
Figure 1: HPE of a TB granuloma (Courtesy: Dr S.Jamuna Rani,
MD (Pathology), Associate professor, Tagore Medical College and
Pathogenesis Hospital, Chennai)
yy Mode of infection is by droplet nuclei (mainly the smallest
droplets <5–10 μm in diameter)
yy After entering the respiratory tract – the risk of developing Clinical Manifestations
disease mainly depends upon individual’s innate immunity yy TB is classified based on the clinical features as pulmonary,
and cell-mediated immunity extrapulmonary or both.
yy Clinical illness that develops first, mainly in children is called yy It classically presents as low grade fever, cough with
primary TB expectorations, weight loss, loss of appetite, hemoptysis,
yy Bacilli that remains in the body for years and being persist – cervical lymph nodes enlargement.
later in life can go for reactivation leading to secondary TB yy Systemic extrapulmonary infections can affect any site and
yy It is estimated that up to 10% of infected people will develop may present as:
TB in their lifetime.  Pleural TB
 Lymphadenopathy
Primary TB  Genitourinary TB
 Skeletal TB
yy It is seen in children; the alveolar macrophages that engulf
 TB meningitis
the TB bacilli make it multiply intracellularly; this leads to
 Tuberculoma
localized subpleural infection affecting lower lobe or lower
 Gastrointestinal TB
part of upper lobe named as Ghon focus
 Pericardial TB
yy Ghon focus along with the hilar lymphadenopathy is called
Primary complex. (Occurs after 1–2 months after infection).
yy Around 2 to 6 months, the lesion heals leaving a calcified
nodule.
Laboratory Diagnosis Liquid media used are Middlebrooks 7H9 media (used in

Chapter 18     Mycobacterium


automated cultures like MGIT)
Sputum Microscopy  Liquid media are also helpful for antimicrobial suscep-
yy Sputum should be collected and concentrated using 4% N tibility testing in Mycobacterial growth indicator tube
acetyl cysteine or 2% NaCl (Petroff’s method) (MGIT)
yy To identify the bacilli in microscopy 104 bacilli/mL should be
present in the sputum; hence concentration techniques will Remember
improve the chance of detection Culture media commonly used are:
yy Ziehl-Neelsen staining method using strong carbol fuchsin •• Lowenstein Jensen media (solid media)
with intermittent heating, followed by 25% H2SO4 (according •• Middle brook 7H10 and 7H11 media (solid media)
to RNTCP) and counterstaining with methylene blue is done. •• Middle brook 7H9 broth (liquid media)

Figure 2: Image of Ziehl Neelsen staining (Courtesy: CDC/ Ronald

W. Smithwic)
 Fluorescent staining using Auramine rhodamine or
acridine orange and counting done by LED microscope is Figure 3: Growth of M.tb colonies in LJ media
most sensitive as it is done in high power field and it covers (Courtesy: Dr Vanathi S, Asst Prof, Dept of Microbiology KAP
many areas Viswanathan medical college, Trichy)

Table 1:  RNTCP grading of tuberculosis smear in AFB

Automated Systems of TB Culture
Number of AFB Grading Number of fields yy BACTEC MGIT
examined yy BACTEC 9000MB
≥10 AFB/ OIF 3+ 20 yy BacT/ALERT
1–10 AFB/OIF 2+ 50
High Yield
10–99 AFB/100 OIF 1+ 100
•• Newer method of cultivation of M.tb  BACTEC MGIT –
1–9 AFB/100 OIF Scanty 100
Automated Mycobacteria growth indicator tube – medium
No AFB Negative 100 used in this system is 7H9 Middlebrook medium with
fluorometric detection based on oxygen consumption by the
Sputum Culture microorganism for its growth
•• MGIT also detects resistance to Pyrazinamide – because this is
High Yield added in the medium

Collection of sputum: (RNTCP) yy Another system used is ESP system – recently this helps to
•• Day 1: Patient should provide on the spot sputum sample detect drug susceptibility of mycobacteria also.
•• Day 2: Patient should bring an early morning sample
Antimicrobial Susceptibility Testing
yy Gold standard for diagnosis of TB is culture which can detect yy Phenotypic methods in LJ media:
even 10–100 bacilli/mL  Absolute concentration method
yy Limitation of culture is generation time for TB bacilli is 14 – 15  Resistance ratio method
hours hence culture takes 1 month to show visible colonies  Proportion method
yy Media used: yy Automated methods
 Solid media like Lowenstein Jensen media, Dorset egg
yy Genotypic methods – detect genes that code for resistance
media, Petragnani medium
(E.g. GeneXpert detects rifampicin resistance)
 Most commonly used in LJ media (sterilized by inspissation
157
as it contains egg)
 Molecular Methods yy Time period to diagnose is less than 2 hours
Unit 2     Bacteriology

yy It helps to identify rifampicin resistance

 Polymerase chain reaction (PCR)
yy WHO and RNTCP recommended diagnostic tool
 Ligase chain reaction (LCR)
 Transcription-mediated amplification (TMA) Table 3:  Ideal method for diagnosis of Tuberculosis
 RFLP
 IS fingerprinting Tuberculosis Methods used to diagnose TB
 Line probe assay Pulmonary TB Sputum culture

Immunodiagnosis TB lymphadenopathy HPE

yy Skin sensitivity testing: TB meningitis CSF culture
 Mantoux test: For screening and to estimate the preva- TB Genitourinary Early morning urine repeated
lence of M.tb collection and culture
 WHO advocates PPD-RT-23 with Tween 80
Disseminated / Miliary Biopsy and culture of bone marrow
 Mantoux test is the m/c used tuberculin test TB and liver tissue and other sites
 Induration should be measured
TB with HIV Sputum microscopy is usually
≤ 5 mm Negative negative; Xpert MTB/RIF assay is
sensitive
6 – 9 mm Equivocal
≥ 10 mm Positive Treatment
 Uses of tuberculin test: For diagnosis of active infection in Table 4:  Treatment as per RNTCP Guidelines
infants and young children; it measures prevalence and
incidence of infection but not the disease First-line drugs Second-line drugs
•• Isoniazid (H) •• Ethionamide
IGRA: Interferon Gamma Release Assays •• Rifampicin (R) •• Thiacetazone (T)
yy It is a test done with whole blood to diagnose Mycobacterium •• Pyrazinamide (Z) •• Para aminosalicylic acid
tuberculosis infection •• Ethambutol (E) •• Bedaquiline
yy They do not differentiate latent TB infection from active •• Streptomycin (S) •• Amikacin
tuberculous disease •• Capreomycin
yy It measures T cell release of IFN gamma •• Cycloserine
yy It is very useful in vaccinated populations as it is not affected
•• Ciproloxacin
by vaccines like it happens in tuberculin test
•• Rifabutin
yy Two tests approved are:
 QuantiFERON – TB Gold test
•• Kanamycin
 T-SPOT: TB test

DOTS–Short Term Regimens

Table 2:  Difference between QuantiFERON TB Gold test yy Rapid response
and T-SPOT yy Lower failure rate
yy Lesser chances for resistance
QFT-GT T-SPOT
yy Better patient compliance
Process whole blood within 16 Process peripheral mononu-
hours clear cells within 8 hours Category I – new HRZE thrice a week for 2 months followed
Antigens used: ESAT-6, CFP-10 Antigens used: ESAT-6 & CFP-10 patients by HR daily or thrice a week for 4 months
& TB7.7 (single mixture of all) (Separate) Category II – HRZES thrice a week for 2 months
Concentration of IFN gamma is Number of IFN gamma Previously treated followed by HRZE for 1 month followed
measured producing cells are measured patients by HRE thrice a week for 5 months

GeneXpert MTB – RIF Assay Drug-Resistant TB

yy It is a molecular rapid test method for the diagnosis of yy DOTS plus refers to the programs that have components for
pulmonary and extrapulmonary TB MDR TB diagnosis and treatment
yy Principle used in Real time PCR yy Proper antimicrobial susceptibility testing should be done
yy It is a cartridge-based assay to coin the term MDR TB or XDR TB

158
Table 5:  Treatment of drug-resistant TB

Chapter 18     Mycobacterium

Definition Treatment
MDR TB : Kanamycin + ofloxacin +
Multi drug resistant TB – Ethionamide + pyrazinamide +
Resistant to rifampicin and ethambutol + cycloserine (6–9
isoniazid months) followed by
XDR TB: Ofloxacin + ethionamide +
Extensively drug resistant ethambutol + cycloserine for 18
TB – resistant to rifampicin, months
isoniazid and any
fluoroquinolones and at least
one injectable second-line
drugs
Figure 4: Cigarbundle appearance of M.leprae (Courtesy: CDC)

High Yield Pathogenesis

•• Newer Drugs for MDR TB: yy Virulence factor of lepra bacilli is – PGL – Phenolic glyco-
ƒƒ Bedaquiline lipid - 1
ƒƒ Delamanid yy Spreads by droplet infection – low pathogenicity but highly
infectious
Immunoprophylaxis yy Incubation period is very long may be up to 2–5 years
yy Disease primarily infects skin, peripheral nerves and nasal
yy BCG vaccine – Bacille Calmette Guerin vaccine
mucosa but any organ can be affected
yy It is developed from live attenuated strain of M. bovis
yy Based on the presentation, leprosy is classified into:
yy Intradermal injection is given at birth; vaccine should not be
 Lepromatous
given to immunosuppressed babies and not be given above
 Tuberculoid
age of 2 years
 Dimorphic
yy It undergoes series of reactions from papule to vesicle finally
 Indeterminate
forming a scar
 Pure neuritic type (seen only in Indian type)
yy The immunity ranges from 0–60% and may last for 10–15 years
yy It mainly protects from miliary and disseminated serious TB
Table 6:  Differences between tuberculoid leprosy and
yy Complication of vaccine:
lepromatous leprosy
 Locally abscess or ulcerations may occur

 Enlargement and suppuration of draining lymph nodes Tuberculoid leprosy Lepromatous leprosy
 Generally fever with mediastinal adenitis may occur
Paucibacillary disease Multibacillary disease
Minimally infectious; less severe Highly infectious
Chemoprophylaxis
yy Izoniazid is given for prophylais in those who has Skin lesions are asymmetric in Symmetrical skin lesions
 Children who have contact with active TB (usually in the distribution
same house) Nerves involved are ulnar, All organs systems are
 Infants of mother with active TB posterior auricular, peroneal and involved except lungs and
 Latent TB posterior tibial nerve CNS
 HIV patients having contact with active TB.
CMI present CMI absent

MYCOBACTERIUM LEPRAE Granulomatous lesions Non granulomatous

yy Acid-fast organism but it differs from M.tb – needs 5% H2SO4 Lepromin test positive Lepromin test negative
in acid-fast staining method (because it is less acid fast than
M.tb)
yy Not alcohol fast Lab Diagnosis
yy Bacilli are seen singly or in groups sometimes the bacilli yy Microscopy is the best method to diagnose in resource-poor
appears grouped together by a lipid-like substance called glia areas:
– this group is called globi;  Slit skin smear technique should be followed or skin
yy Each globus appears as parallel rows and gives cigar-bundle punch biopsy or nerve biopsy specimens can be used
appearance;  Smears are graded based on the number of bacilli 159
 Table 7:  Grading of smears Treatment
Unit 2     Bacteriology

Table 9:  Doses of drugs in multibacillary and

Bacilli per field Grading
paucibacillary leprosy
1 – 10 bacilli in 100 fields 1+
Drug Multibacillary Paucibacillary
1 – 10 bacilli in 10 fields 2+ Rifampicin 600 mg once a month 600 mg once a month
1 – 10 bacilli per field 3+ Dapsone 100 mg daily self- 100 mg daily self-
administered administered
10 -100 bacilli per field 4+
Clofazimine 300 mg once a month
100 – 1000 bacilli per field 5+ or 50 mg daily
>1000 bacilli or clumps and globi in every field 6+ Treatment 12 months 6 months
duration
yy Bacteriological index is calculated by adding all the grading in
all the smear divided by the number of smears ROM regimen: Rifampicin, Ofloxacin and Minocycline
yy Morphological index is calculated as the percentage of solid
fragmented granular bacilli (SFGB) out of the total number High Yield
of bacilli
Lepra Reactions
yy Criteria for calling solid rods are:
•• Type 1: Downgrade and reversal reactions – occurs in border-
 Uniform staining of entire organism
line leprosy; type IV DTH hypersensitivity – neuritis occurs;
 Parallel sides
ƒƒ Treatment is corticosteroids with dapsone or rifampicin
 Rounded ends •• Type 2: Erythema nodosum leprosum – type III hypersensitivity:
 Length 5 times than that of width ƒƒ Treatment is glucocorticoids, thalidominde, clofazimine
yy Methods of cultivation: and chloroquine
 It is not possible to cultivate lepra bacilli in bacteriological
media or in tissue culture
 Generation time: Average: 12–13 days ATYPICAL MYCOBACTERIA
 Foot pad of mice: Intradermal inoculation granuloma yy It is also called as nontuberculous mycobacteria or mycobac-
develops standard procedure for experimental work teria other than tuberculous (MOTT) or anonymous myco-
 Nine banded armadillo: Highly susceptible to infections bacteria
 Artificial culture media: ICRC, Bombay – human fetal
spinal ganglion cell culture, adapted for growth on LJ Table 10:  Classification of atypical mycobacteria
media
yy Test to detect CMI: Lepromin test Runyon classification Species
 0.1 mL of lepromin is injected in the forearm of patient and Photochromogens M. kansasii, M. marinum
observed on 48 hours and 21 days
Scotochromogens M. scrofulaceum, M. gordonae
 Two reactions are noted as follows:
Non-photochromogens M. avium, M. intracellulare,
Table 8:  Early and late reactions in lepromin test M. ulcerans, M. xenopi
Rapid growers M. fortuitum, M. chelonae,
Early reaction Late reaction M. smegmatis, M. phlei
Fernandez reaction Mitsuda reaction
Seen within 24–48 hours Develops after 7–10 days Table 11:  Important points to be remembered
Disappears in 3–4 days after injection and reaches
M. kansasii •• Causes chronic pulmonary disease (DD: TB)
maximum in 3 to 4 weeks
M. marinum •• Fish tank granuloma or swimming pool
Seen as redness and induration Test is read on 21st day
granuloma
in the inoculated site If a nodule of more than 5 mm
If the redness is more than is seen- then test is positive M. scrofulaceum •• Cervical adenitis in children
10 mm – then the test is M. gordonae •• Tap water scotochromogen
positive M. avium •• Called as Battey bacillus
It indicates whether or not a It indicates cell-mediated intracellulare •• Most common NTM isolated from lung disease
person has been previously immunity complex •• Causes pulmonary disease and disseminated
sensitized to lepra bacilli lesions in AIDS patients
It is superior than late reaction M. fortuitum •• Chronic abscess
This reaction is due to DTH to This reaction is due to bacillary M. chelonae •• Injection site abscess
160 soluble constituents of lepra component of antigen M. ulcerans •• Buruli ulcer
bacilli M. vaccae •• Immunomodulator
 MULTIPLE CHOICE QUESTIONS

Chapter 18     Mycobacterium

Mycobacterium Tuberculosis 11. Live TB bacillis culture is by: (Recent Pattern Nov 2015)
a. Tinsdale medium
1. True about M. tuberculosis is: (PGI November 2014)
b. MGIT
a. Strict aerobes b. Gram negative
c. MYPA medium
c. Thin cell wall d. Curved rod
d. BYCE agar
2. Acid fastness of tubercle bacilli is attributed to:
12. GenXpert used for getting diagnosis of TB in:
 (Recent Pattern Dec 2014)
 (Recent PatternJuly 2015)
a. Presence of mycolic acid
a. 1-2 hours
b. Integrity of cell wall
b. 5 hours
c. Both of the above
c. 10 hours
d. None of the above
d. 20 hours
3. Primary complex of M. bovis involves:
13. Fastest method for diagnosis of TB:
 (Recent Pattern Dec 2016)
 (Recent Pattern Dec 2015)
a. Tonsil and lung
a. Gene expert
b. Tonsil and intestine
b. LJ medium
c. Tonsil and skin
c. TB MGIT
d. Skin and intestine
d. BACTEC
4. Which of the following statements regarding Gamma
14. To notify a slide as AFB negative minimum how many
release assays for diagnosis of tuberculosis is true:
fields should be checked: (Recent PatternJuly 2015)
 (Recent Pattern 2012)
a. 20 b. 100
a. First generation Quanti FERON-TB assay used ESAT-6
c. 50 d. 200
b. Second generation Quanti FERON-TB (Gold) assay
15. Radiomimetic BACTEC detect growth of M. tuberculosis
used ESAT-6 and CPF-10
in how much time: (Recent Pattern Dec 2015)
c. These tests can distinguish M.tuberculosis and M.bovis
a. 1 week b. 2-3 weeks
d. None of the non-tuberculosis mycobacteria give a
c. 4-8 weeks d. >10 weeks
positive reaction with the test
16. Tuberculin test is positive if induration is:
5. Reactivation of TB is almost exclusively a disease of-
 (Recent Pattern Dec 2013)
 (Recent Pattern Dec 2014)
a. > 2mm b. > 5mm
a. Bone b. Lymph nodes
c. > 7mm d. > 10mm
c. Lung d. Brain
17. Tuberculin test is: (Recent Pattern Nov 2015)
6. The factor which promotes virulence of M. tuberculosis:
a. Intramuscular b. Intradermal
a. Wax D
c. Subcutaneous d. None
b. Cord factor
18. Negative Mantoux test is when induration is:
c. Muramyl dipeptide
 (Recent PatternJuly 2016)
d. Mycolic acid
a. <5 mm b. <10 mm
7. Rapid examination of tubercle bacilli is possible with:
c. <15 mm d. <20 mm
a. Ziehl-Neelsen stain
19. Positive tuberculin test means:
b. Kin Young stain
 (Recent PatternJuly 2015)
c. Auramine Rhodamine stain
a. Resistance to TB
d. Giemsa stain
b. Susceptibility to TB
8. Mycobacterium tuberculosis grows in LJ media in:
c. Hypersensitivity
 (Recent Pattern Dec 2015)
d. None of the above
a. 10-14 days b. 2-3 weeks
20. Which type of pulmonary TB is most likely to give
c. 4-8 weeks d. >10 weeks
sputum positive: (Recent Pattern Dec 2015)
9. Drug resistance in tuberculosis is due to:
a. Fibronodular
 (Recent Pattern Nov 2014)
b. Pleural effusion
a. Transformation
c. Cavitary
b. Transduction
d. None
c. Conjugation
21. XDR-TB is defined as resistance to:
d. Mutation
 (Recent Pattern Dec 2016)
10. Role of malachite green in LJ medium:
a. INH plus rifampicin
 (Recent Pattern Nov 2015)
b. Fluoroquinolones plus INH plus amikacin
a. Increase growth of M.tuberculosis
c. Fluoroquinolones plus rifampicin plus kanamycin
b. Inhibits growth of other bacteria
d. Fluoroquinolones plus INH plus rifampicin plus
c. Nutritive value
d. As an indicator
amikacin 161
 22. Organism identified by interferons: 34. Mycobacterium leprae can be grown on:
Unit 2     Bacteriology

 (Recent Pattern July 2016)  (Recent Pattern August 2013)

a. Staphylococcus b. Leptospira a. LJ medium
c. Campylobacter d. M. tuberculosis b. Robertson’s cooked meat medium
23. Test done for Mycobacterium tuberculosis by whole c. Foot pad of mice
blood is: (Recent Pattern 2018) d. Sabouraud’s agar
a. IGRA b. BACTEC 35. Hansen’s bacillus is cultured in:
c. Culture d. GenXpert  (Recent Pattern Dec 2016)
a. LJ medium
b. Robertson’s cooked meat medium
Atypical Mycobacteria c. Foot pad of mice
24. True about Mycobacterium other than tuberculosis: d. Sabouraud’s agar
a. Causes disseminated infection 36. True about Mycobacterium leprae:
b. Occurs in persons with normal immunity  (Recent Pattern July 2016)
c. Causes decreased efficacy of BCG due to cross immu- a. Transmitted by droplet infection
nity b. Phenolic glycolipid(PGL) is virulence factor
d. Person to person transmission is seen c. Generation time 12-13 days
25. Pigment producing atypical mycobacteria: d. All are true
 (Recent Pattern Dec 2012) 37. Maximum lepra bacilli are seen in:
a. M. fortuitum and M. chelonae  (Recent Pattern Dec 2015)
b. M. xenopi and MAC a. TT b. LL
c. M. gordonae and M. szulgai c. BT d. BB
d. M. ulcerans 38. ENL is seen in: (Recent Pattern Dec 2012)
26. Which of the following is photochromogenic- a. Lepromatous leprosy
 (Recent Pattern Dec 2014) b. Tuberculoid leprosy
a. M. kansasii b. M. scrofulaceum c. Indeterminate leprosy
c. M. intracellulare d. M. avium d. Pure neuritic leprosy
27. Battey bacillus is: (Recent Pattern July 2015) 39. Urine LAM is used for diagnosis of: (AIIMS Nov 2017)
a. Klebsiella pneumonia a. Mycobacterium tuberculosis
b. Mycobacteria paratuberculosis b. Mycobacterium leprae
c. Klebsiella ozaenae c. Mycoplasma
d. Mycobacteria intracellulare d. Listeria monocytogenes
28. Fish tank granuloma is seen in: 40. Identify the following organism: (AIIMS Nov 2017)
 (Recent Pattern Dec 2014)
a. M. fortuitum b. M. kansasii
c. M. marinum d. M. leprae
29. Buruli ulcer is caused by: (Recent Pattern Dec 2012)
a. Streptococcus b. Spirillium minus
c. M. ulcerans d. Brucella
30. Tuberculosis complex includes all, except:
 (Recent Pattern Nov 2014)
a. M. tuberculosis b. M. bovis
c. M. kansasii d. M. microti
31. Which of the following is not a pathogenic mycobacteria:
 (Recent Pattern Dec 2012)
a. M. kansasii b. M. scrofulaceum
c. M. cheloneae d. M. smegmatis
32. Which of the following is not a rapidly growing atypical
mycobacteria causing lung infection:(AIIMS May 2010)
a. M. chelonae b. M. fortuitum a. Mycobacteria b. Nocardia
c. M. abscessus d. M. kansasii c. Actinomyces d. Corynebacterium
41. Leprosy as a zoonoses is prevalent among: 
M. Leprae  (PGI Nov 2018)
33. Which of the following is true regarding globi in a patient a. Nude mice b. Nine banded armadillo
with lepromatous leprosy: (Recent Pattern July 2015) c. Lion tailed macaques d. Chimpanzee
a. Consists of lipid laden macrophages e. Bats
b. Consists of macrophages filled with AFB
162 c. Consists of neutrophils filled with bacteria
d. Consists of activated lymphocytes
 ANSWERS AND EXPLANATIONS

Chapter 18     Mycobacterium

1. Ans.  (a) Strict aerobes 6. Ans.  (b) Cord factor
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
ogy – 10th ed – Page 351 ogy – 10th ed – Page 354
•• M. tuberculosis is a strict aerobe. •• A factor called cord factor is also responsible for the
•• They are slow growing, nonmotile. Noncapsulated and virulence of M. tuberculosis.
nonsporing •• Mycolic acid is a long chain fatty acid present in the
cellwall of Mycobacteria. It plays a role in pathogenesis
2. Ans.  (a) Presence of mycolic acid and along with peptidoglycan, is responsible for
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- granuloma formation)
ogy – 10th ed – Page 352
7. Ans.  (c) Auramine Rhodamine stain
•• M. tuberculosis resists decolorization with 20% sulphuric
acid when stained with carbolfuchsin by Ziehl-Neelsen Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
method or by fluorescent dyes like auramine O or ogy – 10th ed – Page 358
rhodamine. •• When several smears are to be examined, it is more
•• Acid fastness has been ascribed to the presence of an convenient to use fluorescent microscopy. Smears
unsaponifiable lipid-rich (mycolic acid) wax material in stained with auramine phenol or auramine rhodamine
the cell wall or to a semipermeable membrane around fluorescent dyes and examined under UV illumination.
the cell. •• Bacilli appear as bright rods against a dark background.
Because of the contrast, the bacilli can be seen even
3. Ans.  (b) Tonsil and intestine under high power objective, enabling large areas to be
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- screened rapidly.
ogy – 10th ed – Page 357
8. Ans.  (c) 4-8 weeks
•• M.bovis infection primarily infects cervical and
mesenteric lymphnodes from where it can spread to Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
bone, joints and other extrapulmonary sites. ogy – 10th ed – Page 352
•• Human infection with M.bovis occurs by consuming •• Colonies of M. tuberculosis appears in 2–8 weeks of
raw milk. aerobic incubation at 37 C in LJ media.
•• Negative report should be given only after 12 weeks of
4. Ans.  (b) Second generation Quanti FERON-TB (Gold)
incubation.
assay used ESAT-6 and CPF-10
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- 9. Ans.  (d) Mutation
ogy – 10th ed – Page 258 Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
•• IGRA is a test done in serum ogy – 10th ed – Page 362
•• Helps in diagnosis of latent TB infection
•• Drug resistance in tuberculosis is due to mutations, with
•• Antigens used are: ESAT6, CFP10, TB7.7 of M.tubercu-
losis with an approximate rate of 1 in 108 cell divisions.
•• It measures T cell release of IFN gamma •• Combination therapy can prevent emergence of drug
•• It cannot distinguish between active and latent resistance.
infections
•• It is very useful in vaccinated populations as it is not 10. Ans.  (b) Inhibits growth of other bacteria
affected by vaccinees like of the tuberculin test
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
•• Two tests approved are:QuantiFERON – TB Gold test;
T-SPOT: TB test ogy – 10th ed – Page 352
•• LJ medium is composed of coagulated hen’s eggs,
5. Ans.  (c) Lung mineral salt solution, asparagines and malachite green.
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• Malachite green inhibits the growth of other bacteria
ogy – 10th ed – Page 356 and makes the media selective for M. tuberculosis.
•• Reactivation of latent tubercular infection mainly affects
the upper lobe of the lungs.
•• Lymph node involvement is unusual..

163
 11. Ans.  (b) MGIT 17. Ans.  (b) Intradermal
Unit 2     Bacteriology

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
ogy – 10th ed – Page 353 ogy – 10th ed – Page 360
•• MGIT is Mycobacteria growth indicator tube. •• In mantoux test, 0.1 mL of PPD containing 5 TU is
•• It is a rapid growth detection method, which uses 7H9 injected intradermally (between layers of skin and
Middlebrook medium with fluorometric detection not subcutaneously) on flexor aspect of forearm with a
technology via O2 consumption. tuberculin syringe, raising a wheal.
•• Only live bacteria can consume O2. So the bacilli has to •• The site is examined after 48-72 hours for induration.
be alive for it to be detectable by MGIT.
18. Ans.  (a) <5 mm
12. Ans.  (a) 1-2 hours
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
Ref: tbfacts.org ogy – 10th ed – Page 360
•• Gene expert can directly test sputum samples and give •• Refer to answer no.16.
the results in less than 2 hours.
•• Gene expert test works by detecting DNA in TB bacteria. 19. Ans.  (c) Hypersensitivity
It can also detect the genetic mutations associated with
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
resistance to Rifampicin.
ogy – 10th ed – Page 361
•• It detects both live and dead bacteria. So cannot be used
for monitoring the treatment response. •• A positive tuberculin test indicates hypersensitivity
to tuberculoprotein, denoting infection with tubercle
13. Ans.  (a) GenXpert bacilli or prior immunization with BCG.
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• The test becomes positive 4-6 weeks after infection or
ogy – 10th ed – Page 359 immunization.
•• Tuberculin allergy wanes gradually and disappears after
•• GenXpert results are available in 1-2 hours. 4-5 years in the absence of subsequent contact with
•• The use of liquid media with radiometric growth mycobacteria.
detection such as BACTEC 460 gives results within 2–3 •• In endemic areas, allergy is maintained by repeated
weeks. contacts with the bacilli.
•• MGIT detects bacterial growth about one week prior to
that of LJ medium. 20. Ans.  (c) Cavitary
•• LJ medium detects bacterial growth in 2–8 weeks.
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
14. Ans.  (d) 200 ogy – 10th ed – Page 356 and 357

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• Bacillary shedding in the sputum is higher in cavitation
ogy – 10th ed – Page 358 because the necrotic material breaks out into the airways
leading to expectoration of bacteria laden sputum..
•• When Ziehl-Neelsen staining is performed and sputum •• Bacillary shedding is relatively scanty in lesions that do
smears are examined, a negative report should not be not communicate with the airways.
given till at least 300 fields have been examined, taking
about 10 minutes. 21. Ans.  (d) Fluoroquinolones plus INH plus rifampicin
•• The minimum number of fields to be examined to give a plus amikacin
negative Ziehl Neelsen sputum smear report is 300.
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
15. Ans.  (b) 2-3 weeks ogy – 10th ed – Page 363
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• XDR-TB (Extensively drug resistant MTB) is defined as
ogy – 10th ed – Page 359 MDR-TB that is resistant to isoniazid and rifampicin
plus any fluoroquinolone and atleat one of the three
•• The use of liquid media with radiometric growth
injectable second line drugs (amikacin, kanamycin or
detection such as BACTEC 460 gives results within 2–3
capreomycin).
weeks.

16. Ans.  (d) >10 mm 22. Ans.  (d) M. tuberculosis

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
ogy – 10th ed – Page 360 ogy – 10th ed – Page 360
•• Mantoux or tuberculin test is considered positive if the •• Interferon gamma release assay uses M. tuberculosis
site of injection of PPD shows induration of > or = 10 mm antigen CFP 10 which reacts with T-lymphocytes to
in 48-72 hours. release gamma interferon which can be detected
•• Induration of < or = 5 mm is taken as negative and •• This test is not very specific for pulmonary TB
164 •• It is no longer recommended.
6–9 mm as equivocal.
23. Ans.  (a) IGRA 29. Ans.  (c) M. ulcerans

Chapter 18     Mycobacterium

Ref: Mosby’s manual of diagnostic and laboratory tests – Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
Page 772 ogy – 10th ed – Page 368
•• Interferon-Gamma Release Assays (IGRAs) are whole- •• M. ulcerans causes Buruli ulcer.
blood tests that can aid in diagnosing Mycobacterium •• Ulcers are usually seen in legs or arms and are believed
tuberculosis infection. to follow infection through minor injuries.
•• They do not help differentiate latent tuberculosis •• After incubation period of a few weeks, indurated
infection from tuberculosis disease. nodules appear which break down forming indolent
•• Two IGRAs that have been approved by FDA are ulcers which slowly extend under the skin.
ƒƒ QuantiFERON®-TB Gold In-Tube test (QFT-GIT);
ƒƒ T-SPOT®.TB test (T-Spot) 30. Ans.  (c) M. kansasii
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
24. Ans.  (c) Causes decreased efficacy of BCG due to cross
ogy – 10th ed – Page 351 and 366
immunity
•• M.kansasii is atypical mycobacteria.
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• M.tuberculosis complex includes M.tuberculosis,
ogy – 10th ed – Page 369 M.bovis, M.africanum, M.microti, M.canetti, M.caprae
•• Sensitisation with environmental mycobacteria is and M.pinnipaedi,
believed to interfere with the protective response to
BCG vaccination. 31. Ans.  (d) M. smegmatis
•• This may be one of the reasons for wide variation in Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
protective effect of the vaccine observed in field trials in ogy – 10th ed – Page 368
various parts of the world.
•• M.smegmatis is commonly considered as a saprophyte
25. Ans.  (c) M. gordonae and M. szulgai in smegma but is seldom seen in that location. It is a
frequent isolate from soft tissue infections following
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- trauma or surgery.
ogy – 10th ed – Page 367 •• Other three species given as options are pathogenic.
•• M. gordonae and M.szulgai are scotochromogenic and
form pigmented colonies even in dark. 32. Ans.  (d) M. kansasii
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
26. Ans.  (a) M. kansasii
ogy – 10th ed – Page 366
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• M.kansasii is not a rapid grower but a photochromogen.
ogy – 10th ed – Page 366 •• Other three species given as options are rapid growers.
•• M. kansasii, M. marinum, M. asiaticum and M. simiae
are photochromogenic. 33. Ans.  (b) Consists of macrophages filled with AFB
•• They do not form pigment in the dark, but when young Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
culture is exposed to light for one hour in the presence ogy – 10th ed – Page 371
of air and reincubated for 24-28 hours, a yellow-orange
pigment appears. •• Acid fast lepra bacilli found inside macrophages.
•• The globi appear in Virchow’s lepra cells or foamy cells
27. Ans.  (d) Mycobacteria intracellulare which are large undifferentiated histiocytes.
•• The bacilli are seen as agglomerates being bound
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- together by a lipid like substance known as glia.
ogy – 10th ed – Page 367
•• M. intracellulare is commonly known as the Battey 34. Ans.  (c) Foot pad of mice
bacillus Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
•• It was first identified as a human pathogen at the Battey ogy – 10th ed – Page 372
state hospital for Tuberculosis, Georgia, USA and hence
the name. •• Lepra bacilli are not cultivable in artificial bacteriological
media or tissue culture.
28. Ans.  (c) M. marinum •• Foot pad of mice can be inoculated intradermally with
lepra bacilli. Lepra bacilli could multiply in foot pad of
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- mice when kept at 20°C.
ogy – 10th ed – Page 367
•• M. marinum causes a warty skin lesion called swimming
pool or fish tank granuloma.

165
 35. Ans.  (c) Foot pad of mice •• Crops of tender, inflamed subcutaneous nodules appear
Unit 2     Bacteriology

with fever, lymphadenopathy and arthralgia.

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
•• This is an arthus type response to antigens released from
ogy – 10th ed – Page 372
dead lepra cells.
•• M. leprae is Hansen’s bacillus.
•• Lepra bacilli are not cultivable in artificial bacteriological 39. Ans.  (a) Mycobacterium tuberculosis
media or tissue culture.
Ref: Manual of clinical microbiology – 11th edition – Page
•• Foot pad of mice can be inoculated intradermally with
558
lepra bacilli. Lepra bacilli could multiply in foot pad of
mice when kept at 20°C. •• LAM – Lipoarabinomannan
•• LAM antigen is a lipopolysaccharde present in myco-
36. Ans.  (d) All are true bacterial cell wall–released from metabolically active
cells or degenerating cells with patients having TB
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol-
•• Urine based lateral flow tests are helpful in diagnosis of
ogy – 10th ed – Page 371 and 373
TB especially TB HIV patients
•• The mode of transmission of lepra bacilli may be through
droplet transmission or entry through skin. 40. Ans.  (a) Mycobacteria
•• Phenolic glycolipid (PGL) is a polyketide-derived viru- •• The image shows Acid fast staining with blue background
lence factor produced by Mycobacterium tuberculosis, and clumps of pink bacilli
Mycobacterium leprae, and other mycobacterial patho- •• Bacilli are clustered together and single at some places
gens. •• Acid fastness rules out Actinomyces and Corynebacte-
•• Generation time of M.leprae is 12-13 days on average. rium
•• Nocardia appears as branching filaments, long and
37. Ans.  (b) LL slender
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- •• Hence from the above scenario – this image may be a
ogy – 10th ed – Page 373 culture smear of Mycobacteria – showing many bacilli
•• It may be atypical mycobacteria
•• In LL (lepromatous type) of leprosy, bacilli are seen in •• Only with culture and biochemical reactions we can
large numbers. This is therefore known as multibacillary confirm the organism; this image gives a presumptive
disease. evidence of Mycobacteria
•• Bacilli are shed in large numbers in nasal and oral
secretions. Bacillemia is common. The lepromatous 41. Ans.  (b) Nine banded armadillo
type is more infective than other types.
Ref: https://wwwnc.cdc.gov/eid/article/21/12/15-0501_article
38. Ans.  (a) Lepromatous leprosy •• Nine-banded armadillos (Dasypus novemcinctus) are
Ref: Ananthanarayan and Paniker’s Textbook of Microbiol- naturally infected with Mycobacterium leprae.
ogy – 10th ed – Page 374 •• Hence they are zoonotic transmitters of infection.

•• Erythema nodosum leprosum is type 2 lepra reaction

which occurs in LL and BL types (Lepromatous leprosy),
usually a few months after institution of chemotherapy.

166