MYCOBACTERIUM

Species to be considered:  Due to its cell structure, they become

difficult to stain w/ aniline dyes such as
 Mycobacterium tuberculosis complex Gram stain, but it resist decolorization
 Mycobacterium tuberculosis by acid alcohols after a prolonged or
 Mycobacterium bovis heating w/ a basic fuchsin dye
 Mycobacterium BCG
 They grow more slowly than most other
 Mycobacterium africanum
human pathogenic bacteria because of
 Nontuberculous mycobacteria their hydrophobic cell surface

 Mycobacterium are very thin, rod

shaped and non-motile.
 Have an unusual cell wall structure that
contains N-glycolymuramic acid in lieu
of N-acetylmuramic acid and has a very
high lipid content

Lipid-Rich Cell Wall of Mycobacterium

Mycoli
c acids
Mycobacterium tuberculosis Infections
  • Mixture of protein & fat
(assimilated very
slowly)

 Calcification

• Ca++ salts deposited

 Cavity formation

• Center liquefies &

empties into bronchi

Non-tuberculous Mycobacteria

 NTMs include all other Mycobacterial

species that do not belong to M.
tuberculosis complex
 Present anywhere in the environment
and may colonize healthy individuals in
skin, respiratory and gastrointestinal
tracts

Runyon Classification of NTM

 Attenuated strain of M. bovis, bacille

Calmette-Guerin is used in many parts
of the world to immunize susceptible
individuals against tuberculosis

Typical Progression of Pulmonary Tuberculosis

 Pneumonia

 Granuloma formation with

fibrosis

 Caseous necrosis

• Tissue becomes dry &

amorphous (resembling
cheese)
Non-cultivable NTM – M. leprae
 An NTM that is closely related to M.
tuberculosis
 Called Leprosy or Hansel’s disease
 Leprosy is a chronic disease of skin,
mucous membrane and nerve
tissue
 M. leprae has not been
cultivated in vitro, but can be
grown in armadillo and foot pads
of the mice

Mycobacterium leprae Infections

Tuberculoid vs. Lepromatous Leprosy

Clinical Manifestations and Immunogenicity

Lepromatous vs. Tuberculoid Leprosy


Clinical Progression of Leprosy
TB-DOTS
Directly Observed Treatment Short course
TB-DOTS
 DOTS or Directly Observed
Treatment Short course is the
internationally recommended
strategy for TB control that has
been recognized as a highly
efficient and cost-effective
strategy.
DOTS comprises five components.
1. Sustained political and financial
commitment.
TB can be cured and the epidemic reversed if
adequate resources and administrative support
for TB control are provided.
2. Diagnosis by quality ensured sputum-smear
microscopy.
Chest symptomatics examined this way helps to
reliably find infectious patients.
3. Standardized short-course anti-TB treatment
(SCC) given under direct and supportive
observation (DOT). 
Helps to ensure the right drugs are taken at the
right time for the full duration of treatment.
4. A regular, uninterrupted supply of high
quality anti-TB drugs.
Ensures that a credible national TB programme
does not have to turn anyone away.
5. Standardized recording and reporting
Helps to keep track of each individual patient
and to monitor overall program performance.
The new global Stop TB Strategy builds
on the DOTS strategy, which remains the
fundamental basis for TB control. The six
additional essential elements in the new
strategy are:
1.      Sustaining, improving and
accelerating quality DOTS expansion
2.      Addressing TB-HIV, MDR-TB and
other special challenges
 TB/HIV collaborative
interventions
 DOTS-Plus for MDR-TB
 Reaching vulnerable, high risk
groups
3.      Contributing to health system
strengthening
  TB control innovations that strengthen
health systems
 Adaptation of innovations from other
TB control programmes
 Practical Approach to Lung Health
4.      Engaging all care providers
 Public-private partnerships
 Ensuring equitable access to
international standards of care for TB to
all
5.      Empowering patients and
communities
- Advocacy, communication and
social mobilisation\
- Community based TB care
6.      Enabling and promoting research
 Programme-based operational research
 Development of new diagnostics, drugs
and vaccines
DIRECT SPUTUM SMEAR MICROSCOPY
ROLE OF THE LABORATORY (AFB SMEAR
MICROSCOPY) IN TB CONTROL AND DOTS
 Detection of infectious cases
 Monitoring of treatment progress
 Documentation of cure
Note: Detection and treatment of
infectious cases reduces the spread of
Tuberculosis
PRECAUTIONS IN PROPER COLLECTION OF
SPUTUM
Safe Specimen Collection
 If a patient presents to the laboratory,
and is coughing, ask the patient to
cover his or her mouth
 Collect specimens outside where air
movement will dilute droplet nuclei
and/or sunlight will rapidly inactivate TB
bacilli
 Stand clear of patients when specimens
are collected in TB
 Never stand in front of the patient
during collection
LABORATORY SET-UP
 Ideally, the TB laboratory should be a
well-ventilated area which is dedicated
to microbiology with restricted access.
Three separate areas are recommended
for performing TB microscopy.
1. Smear preparation and staining: This area
should be well lit and preferably near an open
window to ensure adequate ventilation during
smear preparation. A sink with running water is
also required.
2. Performing microscopy: This area should
have a flat bench or table for placement of the
microscope. Subdued lighting is preferred. If
no electricity is available, daylight must be used
as the light source; in this case, place the
microscope directly in front of a window.
3. Record keeping and storage: This third area
is for entering data into the log book for Quality
Control and for storing slides.
COLLECTING SPECIMEN
Sputum collection poses a potential risk in
acquiring TB. It must be done in open air and
at a safe distance from other people.
Never collect sputum in the laboratory!
 Give a new sputum container to each
patient from whom sputum examination for TB
is requested. Demonstrate how to use it to
collect a good specimen. Clearly instruct the
patient on:
STEP 2: dalawang beses na HUMINGA NANG
MALALIM … HUMINGA NG PALABAS
STEP 3: sa ikatlong pagkakataon HUMINGA
nang malalim.. UMUBO nang malakas

STEP 4: IDURA ang plema sa sputum specimen

 the importance of sputum examination
for diagnosis or follow-up of TB;
 how to open and close the containers;
 the need for collecting real sputum, not
saliva;
 how to produce good sputum (i.e., by
repeated deep inhalation and
exhalation of breath followed by cough
from as deep inside the chest as
possible);
 how to avoid contamination of the
exterior of the container (i.e., by
carefully spitting and closing the
container);
 how to collect and safely deliver the
morning sputum to the laboratory; and
 the need for three sputum specimens
to facilitate diagnosis.
 A good specimen should be
approximately 3–5 ml.
 It is usually thick and mucoid, may be
fluid and contain pieces of purulent
material.
 Color varies from opaque white to
green. Bloody specimens will appear
reddish or brown. Clear saliva or nasal
discharge is not suitable as a TB
specimen.
cup.
STEP 5: maingat na PUNASAN ang labas ng
sputum specimen cup, ITAPON ang ginamit na
disposable tissue sa llagyan ng nakahahawang
basura
STEP 6: MAGHUGAS mabuti ng mga kamay
STEP 7: IPAKITA ang plemang nakolekta sa
kawani ng DOTS facility
Optimum Collection Location: Microscopy
Centre
 Specimen is fresh
 Collection supervised
 Immediate recollection, if necessary
Request for Sputum Examination Form Should
Include:
 Patient’s name, sex, age, and address
 Date of collection
 Name of Health Institution
 Reason for examination
Request for Sputum
Examination Form

TAMANG PARAAN NG PAGKOLEKTA NG

PLEMA

STEP 1: MAGMUMOG ng mabuti para tiyakin na

walang matitirang pagkain sa bibig
Labeling Specimen Container

Specimen Receipt at Laboratory

 Check specimens for quality:

 Volume (at least 3–5 ml)
 Describe sputum consistency (mucoid,
purulent, bloody, or watery)
 Register the specimen and allocate a
laboratory serial number  Spread the sputum evenly over the
central area of the slide using a
Specimen Quality
continuous rotational movement

Ziehl-Neelsen method of stain is the standard

procedure for sputum microscopy.

Read at least 300 visual fields before reporting

a negative result. (Note: Fewer than 300,
usually 100 fields may be read if the slide is
found positive for AFB.)
Usually examining 100 visual fields takes about
5 minutes.

Read the slides in a horizontal manner

Purulent Mucoid

APPEARANCE OF AFB IN THE SMEAR

 AFB stain red or pink against a blue
background with the Ziehl-Neelsen
staining method.
 They are usually thin and rod-shaped,
but occasionally may appear as coccoid
(beaded), filamentous (thread-like), or
clumped (in group) forms.
 Why is this smear not acceptable?
 Notice that polymorphs are absent and
numerous squamous epithelial cells are
present. Remember that these are the
features of a smear from a poor quality
specimen or saliva.

AFB - in Various Arrangements

• In this slide , AFB resemble fine red rods

standing out against the blue background. They
may be slightly curved, granular, and may occur
singly, in pairs, or groups. ( POINT OUT the
single AFB in the slide )
 ASK participants if this smear is
acceptable or not. The polymorphs
are numerous and evenly distributed
across the smear. No squamous
epithelial cells are present. These are
the features of a properly prepared
smear from purulent sputum.
 AFB may be arranged in different ways.
( POINT OUT the “V” forms on the slide
and also the single AFBs.)

Microscopy Report
Example
 W
h
e
n
AFBs are present in large numbers,
they can align with one another to form
small clumps or rope like patterns also
known as cords. (POINT OUT the clumps
of tubercle bacilli on the slide.)

AFB Stain reporting

Laboratory Request Form

Example
Microscopy Request and Report Form
Example