2005 L. Ditiu (Bucharest, Romania)
ERS School Courses 2005
Traditional diagnosis and
laboratory organisation
Lucica Ditiu
October 25 – 27, 2005
Bucharest, Romania
2005 L. Ditiu (Bucharest, Romania)
• Direct diagnostic tests – the presence of
the etiological agent is determined
• Indirect diagnostic tests- indicate the
host’s response and indicate the presence
of the disease
• Indirect: clinical symptoms, X-rays,
general laboratory examinations,
immunological assays
2005 L. Ditiu (Bucharest, Romania)
2005 L. Ditiu (Bucharest, Romania)
Bacteriology
Sputum microscopy
Simplest, quickest, most reliable and most economical
- Available
- Cheap
- Rapid
- High predictive value (>90%) the prevalence of
tuberculosis in the population tested is > 10%
- Specificity of 98%
- Correlate with infectiousness
Identification of 70% of bacteriologically positive TB
pulmonary cases
2005 L. Ditiu (Bucharest, Romania)
If the bacilli are evenly dispersed
• The amount of sputum on a slide is 0,01 ml
• At 5,000 bacilli/ml – 50 bacilli on the smear
• 50 bacilli spread on 10,000 fields = 1/200 fields!!!!
• To find 3 bacilli= 600 fields to be examined
• 1bacillus/every 10 fields = 100,000 bacillus/ml
• 1 bacillus/field =1,000,000 bacilli/ml
• A positive smear can be obtained with about
10,000 bacilli//ml
2005 L. Ditiu (Bucharest, Romania)
• High reproductibility of the results – 94%
agreement for smears prepared from the
same specimens and examined
independently
• Smear microscopy performed by non-
specialized health workers may be reliable
if training is provided and performances
continuously monitored
2005 L. Ditiu (Bucharest, Romania)
However,
• Relatively insensitive diagnostic procedure
• Varies: the type of lesion, the type and
number of specimens, the mycobacterial
species, staining technique and the
alertness and persistence of the
microscopist
• A smear positive sample for acid fast bacilli
– either M. tuberculosis, another non
tuberculous mycobacterium
2005 L. Ditiu (Bucharest, Romania)
False positive results
Other acid fast particles - food particles, precipitated stains,
environmental acid-fast bacilli, non- tuberculosis
mycobacteria, Nocardia species, spores of Bacillus
subtilis, yeasts, fibers and pollens
Contamination through transfer of bacilli from one smear to
another
False negative results
Improper sputum collection
Improper storage of sputum and stained smears
Failure to select suitable sputum particles
Improper preparation, staining, examination and reading of
slides
Administrative errors
-Mistakes in labeling, in identification of patients, in code
numbers and specimens
2005 L. Ditiu (Bucharest, Romania)
Fluorescence microscopy (FM)
• Introduced in 1930
Main advantage: the use of a low power objective
(25X) therefore the same area of smear can be
scanned in a shorter time 91-2 min/ 15 min ZN)
• FM in 1 minute gave more true positive results
(and no more false positive) than ZN in 4
minutes
Main disadvantages: the cost of the microscpy unit
and the maintenance, the necessity of standard
electrocal power.
2005 L. Ditiu (Bucharest, Romania)
• 3 sputum specimens
- on spot
- overnight specimen – higher quality
- on spot specimen
2005 L. Ditiu (Bucharest, Romania)
2005 L. Ditiu (Bucharest, Romania)
Culture
• The only definitive diagnosis of tuberculosis–
99% specificity
• Detect lower numbers of AFB/ml (10 -100/ml)
• Makes possible the identification of
mycobacterial species
• Essential role in case classification – non
viable bacillus may remain microscopically
visible even 5 months or more after
treatment
• Adds 20-30% to the total number of
bacteriologically confirmed cases
2005 L. Ditiu (Bucharest, Romania)
•Mycobacterium tuberculosis proliferate extremely
slowly
•The media which allow abundant growth of M.
tuberculosis are egg-enriched media containing
glycerol and asparagine (LJ), and agar/liquid
media supplemented with serum or bovine
albumin
•0.1 ml of a specimen, decontaminated is
inoculated into slopes of the solid media and at
temperature of 35-37 degrees for up to 8 weeks (
2-4 weeks for agar based media)
•Weekly examination to select visible colonies
and to identify contamination
2005 L. Ditiu (Bucharest, Romania)
Disadvantages of culture
• Specially trained and skilled staff
• Long interval before results are available
• Special facilities - permanent supply of
water and electricity
• Costly equipment
• Reliable thermoregulation of hot room
• Biosafety measures
2005 L. Ditiu (Bucharest, Romania)
Liquid media
• Most species grow more quickly
• 1977- Middlebrock – detects growth of
mycobacteria based on the release of the
radiolabelled C02 (BACTEC) – 7-14 days
detection time
• Allows DST to be performed in the same
time as well as the identification
• Disadvantage: cost of the method
2005 L. Ditiu (Bucharest, Romania)
Smear positive, culture negative
• Treatment
• Sputum specimens: exposed to sun light,
heat, stored too long, dried out,
contaminated.
• Excessive decontamination, over-heating
during centrifugation, inadequate culture
media, deficient incubation
• Non tuberculous mycobacteria
2005 L. Ditiu (Bucharest, Romania)
Use of culture
• Gold standard - confirm the TB diagnosis
• Diagnosis of cases with clinical and radiological
signs of pulmonary tuberculosis where smears are
repeatedly negative
• Diagnosis of extra-pulmonary and childhood
tuberculosis
• Diagnosis in HIV/TB patients
• Follow-up of tuberculosis cases who fail a
standardised course of treatment and who may be
at risk of harbouring drug resistant organisms
• Surveillance of tuberculosis drug resistance as an
integral part of the evaluation of control
programme performance
• Investigation of high-risk individuals who are
symptomatic
2005 L. Ditiu (Bucharest, Romania)
Radiology
• No radiographic pattern is diagnostic of
TB!
• The chest radiography is unrealiable as
unique tool in the diagnosis or follow up of
TB cases!
• Unreliable: observers errors (inter and
intra observer variation), over and under-
reading, influence of experience.
• Atypical radiographic pattern seen in
patients with TB/HIV
2005 L. Ditiu (Bucharest, Romania)
Imunological tests
• The tuberculin skin test – can not differentiate between latent and
active disease. Tool available for diagnosis of TB infection
• Serology –blood tests to measure the humoral response to M.
tuberculosis
– Has low sensibility and sensitivity – the primary response is cell
mediated
– Sensitivity is high in smear positive disease but is low in children, extra-
pulmonary TB, TB/HIV
• Cell mediated immunity – circulating lymphocytes are extracted
from the venous blood and exposed to antigens of M. tuberculosis
and after 6-24 hrs the production of inflammatory mediators -
interferon gamma is measured.
– Measures the primary immune response of humans to TB
2005 L. Ditiu (Bucharest, Romania)
Laboratory network
• A structure in which various laboratories
working at different levels of service
complexity are linked by the common
objectives of the NTP.
• Common sets of standards, information
systems, materials and services offered,
quality assurance.
T. Chonde, R. Cruz, I.deKantor, A.Laszlo, N.K.Jain, O.Latini, R. Rodrigues,
P.Wright, R. Urbanczik - International course on management of TB
laboratory networks in low income countries: International Union Against TB
and Lung Diseases, World Health Organization, Health Canada, 2000
2005 L. Ditiu (Bucharest, Romania)
Bacteriological laboratory is responsible for
confirming the disease!
• Accessible, timely and reliable:
- the lab should be as close as possible to the place
where the patient received medical care or existence
of sputum specimen transportation
- Take the specimen on request and deliver results on
timely fashion
- Have the resources to carry out the requested test
- Ensure the highest possible quality
DIFICULT TO BE MET BY AN ISOLATE LABORATORY
2005 L. Ditiu (Bucharest, Romania)
Levels of laboratory services
• The peripheral (often district) laboratory
– sputum smear microscopy Ziehl-Neelsen (ZN) staining of
unconcentrated sputum specimens
• The intermediate (often regional) laboratory
– sputum smear microscopy Ziehl Nielsen, fluorochrome staining,
cultures, identification
– provide supervision, monitoring, training and quality assurance
to peripheral laboratories
• The central (often national) laboratory
– capable of performing microscopy (both ZN and fluorescence),
mycobacterial culture, drug susceptibility testing and species
identification
– training for laboratory staff, perform quality assurance and
proficiency testing, exercise surveillance of primary and acquired
tuberculosis drug resistance and participate in epidemiological
and operational research.
2005 L. Ditiu (Bucharest, Romania)
Development of a laboratory service for
TB in a high prevalence country
• Establishment of ZN microscopy in small,
multi-purpose public health laboratories
– The maximum number of ZN smears examined per
microscopist per day should not exceed 20.
– proficiency in reading ZN smears can only be maintained
by examining at least 10 to 15 ZN smears per week, ie. a
minimum of 2-3 examinations per day
• Establishment of fluorescence microscopy
at regional laboratories
– more than 100 smears are examined per day
– one fluorescent microscopy centre per 500 000 to one
million population is usually sufficient
2005 L. Ditiu (Bucharest, Romania)
Development of a laboratory service for
TB in a high prevalence country
• Establishment of tuberculosis culture
facilities at regional or central level
• to cover 500 000 to one million population
• Establishment of a central reference
laboratory at national or regional level,
• to cover 10 million or more population
2005 L. Ditiu (Bucharest, Romania)
PERIPHERAL LEVEL
• Technical
– preparation and staining of smears
– ZN microscopy and recording of results
– internal quality control
• Administrative
– receipt of specimens and despatch of results
– cleaning and maintenance of equipment
– maintenance of laboratory register; reporting
– management of reagents and laboratory supplies
2005 L. Ditiu (Bucharest, Romania)
INTERMEDIATE LEVEL
All the functions of the peripheral level, plus:
• Technical
– fluorescence microscopy (optional)
– digestion and decontamination of specimens
– culture and identification of M. tuberculosis
– preparation and distribution of reagents for
microscopy in peripheral laboratories
• Managerial
– training of microscopists
– support to and supervision of peripheral staff with
respect to microscopy
– external quality improvement and proficiency testing
of microscopy at peripheral laboratories
2005 L. Ditiu (Bucharest, Romania)
CENTRAL LEVEL
All the functions of the intermediate level, plus:
• Technical
– drug susceptibility testing of M. tuberculosis isolates
– identification of mycobacteria other than M. tuberculosis
• Administrative
– technical control of and repair services for laboratory equipment
– updating and dissemination of manuals on bacteriological
methods for diagnosing tuberculosis
– development and dissemination of guidelines on care and
maintenance of microscopes and other equipment used in
tuberculosis bacteriology
– development and dissemination of guidelines on tuberculosis
laboratory supervision and quality assurance
– collaboration with the central level of the National Tuberculosis
Programme in defining technical specifications for equipment,
reagents and other materials used in bacteriological
investigations, and in estimating laboratory materials and
2005 L. for the Programme budget
equipment requirements Ditiu (Bucharest, Romania)
CENTRAL LEVEL (cont.)
• Managerial
– training of intermediate laboratory staff in bacteriological
techniques and support activities, ie. training, supervision, quality
assurance, safety measures and equipment maintenance
– supervision of intermediate laboratories regarding bacteriological
methods and their support (particularly training and supervision)
to the peripheral laboratories
– quality assurance of microscopy and culture performed at
intermediate laboratories
• Research and surveillance
– organization of surveillance of primary and acquired
mycobacterial drug resistance
– operational and applied research relating to the laboratory
network, co-ordinated with the requirements and needs of
National Tuberculosis Programmes.
2005 L. Ditiu (Bucharest, Romania)
Staff needs
• Peripheral level – one technician or auxiliary
technician, according to the workload
• Intermediate level – a senior bacteriology
technician with post secondary or university
training; technicians, maintenance staff
• Central level – a bacteriologist in charge (university
trained) senior technicians, administrative assistant,
laboratory assistants, maintenance staff
• National Reference Laboratory: head of the
laboratory, university trained bacteriologists,
technicians, statisticians, administrative and
maintenance staff.
2005 L. Ditiu (Bucharest, Romania)
New developments
Global Plan 2006-2015 STOP TB
By 2006 By 2010 By 2015
vaccines 3 vaccines in phase 9 candidates in 4 phase III trials
I trials phase II trials; at carried out; one
least 2 vaccines in safe, effective
"proof of concept" vaccine available
trials; beginning
phase III trials
drugs 27 new compound 1-2 new drugs 7 new drugs;
sin the pipeline registered; treatment shortened
treatment shortened to 1-2 months
to 3-4 months
diagnostics rapid culture for point of care, rapid predictive test for
case detection and culture, improved LTBI
DST in microscopy, phage
demonstration detection, simplified
phase NAAT
2005 L. Ditiu (Bucharest, Romania)