Lesson № 1.

Method of inspection of patient with tuberculosis

WHO Guidelines for treatment of tuberculosis

fourth edition
http://www.who.int/tb/publications/2010/9789241547833/en/

who guidelines for the programmatic management of drug-resistant tuberculosis

2011 update
http://whqlibdoc.who.int/publications/2011/9789241501583_eng.pdf
General approaches to diagnostics of tuberculosis.
Special methods of exposure and diagnostics of tuberculosis
(microbiological diagnostics,X-Ray diagnostics, tuberculinodiagnostics).
Curation of patients.

General approaches to diagnostics of tuberculosis.

Clinical examination of tuberculosis patients

The methods of investigation of respiratory (tuberculosis) patients are

conveniently divided into three groups.
The First group – compulsory (obligatory) methods, which embrace clinical
examination of a patient (complaints, anamnesis, examination, palpation,
percussion, auscultation), thermometry, X-ray investigation (fluorography, X-
raygraphy, X-rayscopy), sputum analysis for MBT, Mantoux tuberculin test (with 2
TU), general blood and urine test.
The Second group – additional (supplementary) methods, which include
repeated sputum analysis (bronchial lavage water) for MBT, tomography of the
lungs and mediastinum, protein-tuberculin tests, immunologic tests, instrumental
examinations (bronchoscopy, biopsy, bronchography, pleuroscopy).
The Third group – facultative (optional) methods: investigation of the outer
breathing function, blood circulation, liver and other organs and systems.
Tuberculosis is an infectious disease, caused by MBT, and is characterized
by the development of specific inflammation in injured organs and polymorphism
of clinical symptoms – intoxication and local syndromes.
 Within the range of the likely evidences of the general TB intoxication, the
most frequent are general weakness, indisposition, drop of the ability to work,
sweating, appetite drop, weight loss, sleep disturbance, body temperature rise.
Body temperature in TB patients can vary – it can be normal, subfebrile, febrile
and even hectic. However, the most often it is subfebrile, which is characterized
with pronounced lability and the absence of monotony. Patients often endure the
high temperature easily.
At the beginning of the illness the sweating is low. Profuse sweat, mainly at
night, are characteristic of pronounced exudative, caseous specific processes.
The local manifestations of lung tuberculosis are: prolonged cough, sputum
secretion, haemophthisis, chest pain, shortness of breath.
Cough is the most frequent symptom in patients with lung TB. At the initial
stages of the process the cough is quiet, not frequent, in the form of light constant
hacking cough. Persistent convulsive loud cough is characteristic for patients with
tuberculous bronchoadenitis and tuberculous endobronchitis.
At the beginning of the illness, the sputum can not be secreted or can be
secreted only a bit. With the progress of the tuberculous, in particular the
distructive, process, the patient may secret up to 200 ml of the sputum, which can
be mucous or mucous-purulent and can be without any unpleasant smell.
Chest pain is often present already at the beginning of the illness , which is
caused by the extensive process in the lungs. Acute sudden pain appears at
spontaneous pneumothorax.
Dyspnea is not characteristic for the initial forms of TB, except for the
miliary TB and the exudative pleurisy. At the chronic extensive process,
complicated with breathing or pneumo-cardial insufficiency, dyspnea can be
brightly expressive.
Hemoptysis and bleeding may be present at any form and phase of the
process, but more often – at destructive forms of TB, and more rarely – at
posttuberculous pneumosclerosis with bronchoectasia. Hemoptysis is characterized
by the presence of veins, blood admixtures in the sputum and of separate blood
spits. At pulmonary bleeding, much more of pure blood is coughed up in one time
(over 10 ml), continuously or with breaks. Blood is usually bright-red, foamy, with
tiny air bubbles; it doesn’t have tendency to coagulation. After the bleeding or
hemoptysis stops, blood clots are coughed up for several days more; and due to the
blood aspiration, the boby temperature rises.
The beginning of tuberculosis illness may be without any symptoms,
subacute and rarely – acute. While interviewing a patient it is very important to
find out a fact about his contacts with tuberculosis patients. To the point, persons,
living amidst nidi of tuberculosis infection (the patient, suffering from the
tuberculosis, who excretes the mycobacteria, the place where he lives and people,
who live with him), 5-10 times more often contacting tuberculosis. Of great
importance is the information about endured in the past “flu”, pneumonia,
exudative pleurisy, under which mass tuberculosis can run; accompanying
illnesses, working conditions, harmful habits etc.
The formal examination at initial forms of tuberculosis reveals no patient’s
visible abnormalities. However, in most patients, at the more advanced stages of
the disease one can find manifestations of tuberculosis intoxication: eyes lustre,
hectic blush on the background of a face pale skin; paraspecific tuberculosis
manifestations (knotty erythema, keratoconjunctivitis, phlyctenae) enlarged
peripheral lymphatic nodes, fistulas or scars after them, chest deformation.
Palpably: often lowered skin turgor, muscles tone, micropolyadenite, positive
“fork-shaped” symptom (putting 2 fingers above the sternum from the both sides
of trahea, it is possible to feel its dislocation to the side of injury), which is
observed at unilateral lung cirrhosis, atelectasis. Increased voice tremor above
infiltration or cirrhosis zones, weakened – at exudative pleurisy, pneumothorax.
Percussively: shortened and dull percussion note is defined above the airless
lung tissue or in the spheres of its lowered (weakened) pneumatization at
infiltrates, nidus-fibrous transmutations (changes) and exudative pleurisy.
Tympanic note occurs above the strenuous spontaneous pneumothorax, a gigantic
cavity. However, more often shortening of percussion note is observed above the
cavern.
The standing hight of the lungs apex and the width of Crening’s spheres
(fields) decreases as a result of nidus, infiltrating or fibrous changes in the upper
sections of the lungs.
Auscultation should be performed consistently above the symmetrical
sections of the lungs at quiet deep breathing with the patient’s half-opened mouth.
With a view to provoke crepitation, the patient should be asked to cough slightly at
the end of the expiration. Herewith the doctor should stay at the patient’s side to
avoid infestation.
It is necessary to find out the type of breathing (vesicular, bronchial, mixed)
and additional murmurs (moist, dry rales, crepitation, pleura friction murmur).
Weakened vesicular breathing is defined at lung emphysema, exudative pleurisy,
pneumothorax and high caloric diet; strengthened – at emaciation, cirrhosis and
lung infiltration process. Rough or bronchial breathing may be heard above a
thickened lung tissue (infiltrate, cirrhosis, fibrosis), amphoric respiration – above a
large cavern with fibrous walls and a wide draining bronchus. Important from the
diagnostic point of view are local moist rales, which are auscultated after the
hacking in the “alarm zones”: frontally, above and beneath the clavicle, from the
behind above the lungs, near a scapula spine and between scapulae. Local small-
bladdered moisty rales are the indication of the beginning of lung tissue
destruction, while those of medium-and-great-bladdered – the indication of a
cavern. In addition to this, medium-or-great-bladdered rales above the upper
sections of the lungs is an essential indication of the decay cavity. Dry rales occur
at bronchitis, whistling ones – at bronchitis with a bronchospasm. At dry
(fibrinous) pleurisy the murmur of pleural rub is heard.
Is directed for prevention infection of healthy people from ill on tuberculosis
people and animals. Infections agent of tuberculosis importance of infection, its
resourse and ways of transmission – there are the base of study.
Sanitary prophylaxis includes sanitary locus of tuberculosis infection, sanitary and
vet care, and early revealing, isolation and treatment of ill on tuberculosis.
Epidemioligical locus includes ill persons, who discharge micobacteria,
house, where ill person lives and people with whom he lives. Bacteriodischarger is
a person, who discharge MBT with a help of any methods of trials and clinico-
radiological features of active tuberculosis process.
Disinfection , test of persons, who were in contact with ill,
chemoprophylaxis, isolation of children from bacteriodischarger, sanitation up
bringing of ill, improvement conditions of living standarts and also treatment of
desease are the most epidemiological factors of tuberculosis infection.
Prophylaxis is performed because epidemic danger, including such factors :
1) spreading of bacteriodischarge
2) when there are children and tecnagers in the family
3) sanitary conditions in which ill-person is living.
The most important factor is spreading of bacteriodischarge, which is
devided into:
1) greate, when micobacteria in spit is present in simple bacteria scopy
2) small, which includes methods sowing
3) formal, when bacteriodischarge stops.
Focus of tuberculosis infection is devided into 3 groups.
1-group: focus, where ill person with big or small bacteriodischarge live, but there
are children and tecnagers in the family, which live poorly.
2-group:ill person with small bacteriodischarge and all members of the family are
adults (without children) or illwwith formal bacteriodischarge and children are in
the family.
3-group:ill person with formal discharge and in the family live only adults.

Early detected of tuberculosis

The sick men on tuberculosis may be detected:
- timely
- intimely
- lately.
The methods of detected the tuberculosis in time are the following ones:
1) prophylactic inspection:
- children – tuberculine diagnostic
- teenagers – tuberculine diagnostic, in 15 years old in addition
fluorography
- adults - fluorography
2) detected in case of appealing for medical help
3) watching for the persons with high risk to become ill in tuberculosis.
The causes of tuberculosis intimely detected are the following ones:
- non-attentive attitude patient to his health
- peculiarities of tuberculosis course
- mistakes of doctors.

Special methods of exposure and diagnostics of tuberculosis (microbiological

diagnostics,X-Ray diagnostics tuberculinodiagnostics).
 The laboratory diagnostics of tuberculosis. Methods of revealing
mycobacterium of tuberculosis. Atipical MBT. Sensitivity of MBT

The source of infestation of human beings are tuberculosis human patients

and animals secreting tuberculosis mycobacteria. The material for revealing MBT
are sputum, bronchial lavage waters, faeces, urine, fistula pus (matter), pleural
cavity exudate, spinal fluid, punctates and bioptates of various organs and tissues.
Sputum examination for MBT is of great epidemiological and clinical
importance. When there is no sputum or it is scarce, expectorants, irritant aerosol
inhalations, bronchi lavage are administered (fig.1).

Methods of Revealing Mycobacteria:

1. Bacterioscopy is one of the main methods of revealing MBT; it includes

ordinary bacterioscopy, flotation and luminescent microscopy. An ordinary
bacterioscopy is accessible to all, simple and quick to do. In smears, coloured
according to Tsil-Nilsen, MBT are revealed when not less than 50000 microbic
bodies are present in 1 ml of pathologic material. According to the “instructions for
bacteriologic diagnosis of the tuberculous infection” of the Ministry of Health
Protection of Ukraine, order № 45 dtd. 06.02.2002, MBT can be revealed if their
quantity is 5000 – 10000 bacterial cells in 1ml of the pathologic material. Under
the microscope MBT look like bacilli of the red colour on the blue background.
 Flotation method (enrichment or concentration of MBT in a small volume,
caused by droplets of benzine, benzole, xylol or toluene on the surface of a retort
ring) is applied in cases when there is a small number of MBT in the pathologic
material and at negative results of ordinary bacterioscopy. Flotation method
provides for 10-15 % more often revealing MBT in comparison to direct
bacterioscopy.
Luminescent microscopy is based on the ability of MBT, coloured with
fluorochroms, to illuminate under the influence of ultraviolet rays and performing
microscopy at small magnification, increasing by 15-30 % the sensitivity of the
method in comparison to direct bacterioscopy and by 10 % in comparison to the
flotation method (fig.2).

2. Bacteriological method consists in the following: sputum or another

material, after preliminary special treatment, is sown on nutritive media (hard,
blood, semisynthetic) (fig. 3). More often hard egg Lewenstein-Yensen medium is
used. 20-100 microbial bodies in 1 ml of sputum is enough for revealing MBT
culture. The first colonies appear on the 14-30-th day of cultivation. The negative
result is given only in 2,5-3 months from sowing. This method of revealing MBT
allows to define their vitality, virulence, group (differentiate from acid resistant
saprophytes and atypical MBT) and species origin, as well as their resistance to
antimycobacterial preparations. In addition to this, according to the data of
bacteriological examination quantitative essessment of bacterial secretion is made:
miserly-up to 20 colonies on a nutrient medium, moderate – from 20 to 100 and
massive – more than 100 colonies.
 Fig. 3. Culture of mycobacteria tuberculosis at hard egg medium

Therefore, the culture result must depict not only the qualitative
characteristics (positive or negative), but also a quantative evaluation (colonies №).
For that it is recommended to use Table 1 (National Antituberculous Programme,
2000).

Table 1
WHO scheme of the evaluation of the results of cultural examination for
M.tuberculosis

Colonies quantity Result evaluation Characteristics

1-19 colonies Positive Individual colonies (miser bacterial
emision); bacterioscopy of the
 pathologic material scarcely reveal
individual mycobacteria
20-100 colonies 1+ Moderate bacterial emission;
bacterioscopy of the pathologic
material reveal individual
mycobacteria in each field of vision
or only single ones – in the
preparation, but not less than five
100-200 colonies 2+ Massive bacterial emission;
200-500 colonies 3+ bacterioscopically – 10 and more
(almost universal mycobacteria in each field of vision
growth) 4+

Over 500 colonies

(universal growth)
Germination of Germination –
common microflora repeat inoculation

3. Biological method. It is infection with sputum or another pathologic

material of guinea-pigs, which are highly sensitive to MBT. A biological testing is
the most sensitive method of revealing MBT, so far as in laboratory animals
tuberculosis develops after the introduction of the material in which there may be
less than 5 microbial bodies in 1 ml. However, it should be noted that MBT are
stable to chemical preparations, particularly to isoniazidum, avirulent for
guineapigs. That is why various methods of microbiological examination should be
used for revealing MBT in pathological material. Generally, before starting to treat
a patient, he should undergo complex bacteriological examination: three times
direct bacterioscopy of sputum or, when it is absent, three times examination of the
material after provoking inhalations or bronchial rinsing waters; with negative
results – three times examination by flotation method; three times sputum sowing
on a nutritive medium, irrespective of the results of the previous examinations,
with a view to define MBT sensitivity to antimycobacterial preparations the
examinations are made in accordance with the recommendations of the World
Health Organization.
The repeatedness of the patient examination for TB revealed for the first
time in the dynamics of chemotherapy (acc. To WHO recommendations) is given
in Table 2.
Table 2
The repeatedness of the patient examination for TB revealed for the first time in
the dynamics of chemotherapy

Period from the first Obligatory minimum

of therapy (month)
Determination of drug
Bacterioscopy Inoculation
resistance

0 х3 х3 х3

At the end 2 (3) х2 х1 х1

From the first

х2 х1 х1
5

At the end 6 (8) х2 х1 х1

Notes:
x
if at the end of the second month of the treatment the results of
bacterioscopy of MBT smear is (+), it’s necessary to make bacterioscopy at the end
of the third month;
xx
if at the end of the sixth month of the treatment the results of
bacterioscopy of MBT smear is (+), it’s necessary to make bacterioscopy at the end
of the eighth month.
 The usage of antimycobacterial preparations provokes the development of
drug resistance to MBT. There are primary and secondary drug resistance.
The primary drug resistance – is MBT stability, in the patients who were TB
revealed for the first time; this stability is the result of the infection with stable
MBT strains.
The secondary drug resistance appears in the process of continuous irrational
antimycobacterial therapy.
According to WHO data, the frequency of the primary drug resistance to any
preparation is 10.4% average, and the secondary – 36 %. In Ukraine the primary
drug resistance twice exceeds the average WHO index, and the secondary – 1,5
times.
Nowadays immunologic and molecular-genetic methods are applied for
etiological identification of tuberculosis. The most promising of them are
immunoenzymic and radioimmune methods of defining mycobacterial antigens
which are based on the application of monoclonal antibodies. Molecular-genetic
method of polymeraze chain reaction consists in revealing in biological material
(sputum, tissues; lavage, pleural, spinal fluid etc.) of mycobacterial DNA. The
examination results may be obtained in 3-4 hours.
The determination of MBT sensitivity to antimycobacterial preparations is of
paramount importance for the treatment tactics, correction of antimycobacterial
therapy and the illness prognosis. MBT sensitivity to antituberculous preparations
is defined by the preparation minimum concentration, which inhibits MBT growth
on the nutritive medium. MBT are considered to be sensitive to either preparation
if less than 20 colonies have grown in a test-tube, with abundant growth in the
control. The culture is supposed to be stable if more than 20 colonies have grown.
MBT are considered to be stable if they grow at the concentrations of the
preparation in 1 ml of the nutrient medium: for isoniazidum – 1mkg, rifampicinum
– 20 mkg, streptomycini – 5 mkg, ethambutolum – 2 mkg, all other preparations –
30 mkg.
 Blood examination. The main reasons of changes in peripheral blood of
tuberculosis patients are intoxication and hypoxia. At initial forms of tuberculosis a
hemogram is normal or with inconsiderable deviations. In recent years
hypochromic anemia is more and more often observed, however at chronic
lingering course of the spread specific process, with the intensification of the
phenomena of lung insufficiency and hypoxia, compensatory increase of
erythrocytes number and hemoglobin is possible. In general, for more serious
clinical forms of tuberculosis slight leucocitosis (9,0-15,0 х 109/l) is characteristic,
percentage of bacillarnuclear neutrophiles, lymphopenia, monocytosis, eosinopenia
increase, ESR is speeded up, as well as the decrease of albumin-globulin
coefficient sets in with the relative increase of alpha-2 and gammaglobulins.
Urine examination. In patients with expressed tuberculosis intoxication,
proteinuria, erytrocytes and leucocytes in urine may be observed. With intoxication
decrease the pathological changes in urine disappear. However, if there is
amiloidosis of internal organs, in particular kidneys, hypoisosthenuria is typical,
stable proteinuria, increased number of erythrocytes, leucocytes in urine, as well as
emergence of cylinders.

DIAGNOSTIC LABORATORY METHODS FOR TUBERCULOSIS

Introduction

In mycobacteriology labs, we continue to use mainly the old, proven

methods. This is so even in rich countries, where it would be less of a problem to
use the newer and more expensive or technically demanding techniques. Smear
microscopy, for instance, a technique which is over 100 years old, continues to be
included in every investigation for TB, simply because it is all over the world a
valuable technique which is helpful in many cases. Because of the higher
proportion of smear-positive cases and the rarity of other mycobacterial pathogens,
this is even more true in TB high-prevalence countries. The reliance on
microscopy for TB in these countries is not in the first place because of lack of
money for something better. Newer techniques exist, but some of them have
serious shortcomings, while others have not yet been evaluated sufficiently to
decide on when and where to use them.

In this module we will briefly discuss the main techniques for detecting MTB.
This implies examining not only test characteristics such as sensitivity and
specificity, but also other factors such as technical requirements and costs.
The diagram below illustrates the various characteristics used to describe a
diagnostic test.

FP = false positives of the test

FN = false negatives of the test

This cross-table shows the usual way to calculate the different values from results
that were obtained comparing a given test against a “gold standard,” which is
believed to indicate the total actually sick ("Disease present") and the total not sick
("Disease absent"). In TB research, the gold standard will usually be culture. For
microscopy rechecking quality control, for instance, it will be the results of the
controllers.
Sensitivity and specificity are the basic characteristics, inherent to the technique
but independent of the population in which it is used. Predictive values do take into
account not only technique, but also the population in which it is used via the
"prevalence" parameter. Prevalence indicates the frequency of the disease and will
be different for different populations. With high prevalence, sensitivity needs to be
high to reach a good negative predictive value (NPV). With low prevalence, so
when the disease is rare, specificity needs to be very high, otherwise the positive
predictive value (PPV) of a test will be poor. This is illustrated in the next
diagram, showing the outcome of the same test (same hypothetical sensitivity and
specificity) applied in populations with highly different prevalence of HIV
seropositivity. PPV as well as NPV are dramatically different. In the first
situation, a negative result still leaves about one chance in four that the patient is
seropositive; in the second, there is less than one percent chance that a positive
result truly indicates seropositivity, despite a seemingly high specificity.

The performance of various laboratory methods for detecting MTB is largely

dependent on the numbers of TB bacilli or their products present in the samples.
All tests will score better on specimens that are rich in bacilli, but only the most
sensitive ones will also detect MTB in paucibacillary disease. As discussed earlier,
the lungs of patients with infectious disease harbor many bacilli, and in high-
prevalence areas these are also the most common type of patients. The curve
below shows what this means for some tests in terms of proportions of patients that
can be detected. AFB-microscopy needs minimally +/-5000 AFB per ml of
sputum to yield a consistently positive result, and this is the case for most patients
who present because of symptoms. This also shows that a distinct difference exists
in sensitivity between excellent and poor AFB-microscopy. Culture as well as
PCR can detect as few as 100 TB bacilli per ml, but this represents fewer patients
in low-income countries. X-Ray is shown for comparison; it can in principle
detect patients who do not excrete any TB bacilli at all.
Review of main lab methods

Roughly, the various tests we use can be classified as tests for detecting whole
bacteria, or as those that detect compounds or products of the bacteria, and those
that detect changes in the patient’s immunologic response to TB bacilli (serology).

1. Detection of whole bacteria

 microscopy for AFB

This will be discussed in detail in a separate chapter.

 culture of mycobacteria
Culture remains the gold standard in mycobacteriology because of its high
sensitivity as well as specificity. Sensitivity reaches a minimum of about 100
bacilli per ml of sputum. In high-prevalence countries this may correspond to
80-90% of patients presenting because of symptoms. Sensitivity for cultures
seems to be lower in AIDS patients with advanced immune deficiency, where
fewer TB bacilli are present in sputum. Sensitivity will also be affected by
technical factors, such as centrifugation and decontamination efficiency. In
terms of specificity, culture has the big advantage by allowing differentiation
between MTB and other mycobacteria, but this is less important in TB high-
burden countries. On the other hand, specificity can suffer with careless
technique. Transfer of TB-bacilli from positive samples may occur more
frequently than when using microscopy. Specificity (assuming properly
executed culture technique) will be around 99%, but in practice it will regularly
be less.
 The main disadvantage of culture is not the need for more sophisticated
resources and more qualified technicians or higher cost, but its slowness. This is
certainly so with the classical egg- or agar-based media (Löwenstein-Jensen,
Ogawa, Middlebrook), for which a positive result requires on the average 3
weeks. The commercial liquid systems (BACTEC, MGIT) may reduce this time
by 50% or more, but they are more costly and place higher demands on
equipment, so that they cannot be routinely used in resource-poor countries.
The diagnostic delay of culture, together with the presence of advanced disease
in many cases at presentation and the high index of suspicion among clinicians
in countries with a high prevalence of TB, all work to reduce the usefulness of
culture under these conditions. It has been shown that, under these
circumstances, three smear examinations yield about the same as one culture. In
addition, physicians will not wait for the results of culture to initiate TB
treatment on other evidence. Moreover, repeating AFB smears after a few
weeks in case symptoms persist, as is generally recommended, may give the
same information as culture.
The conclusion may be that establishing culture facilities outside reference
laboratories should not be a priority in national programs until it becomes clear
that control of the disease is well under way and resources permit. Often these
conditions will coincide, with earlier patient presentation because of economical
improvement, and setting up culture facilities will automatically be part of the
technological progress in general.
For reference laboratories in high-burden countries, culture will mainly serve as
a first step in drug susceptibility testing (DST). DST itself will be reserved to
track drug resistance. In these laboratories, culture may also enhance research.

2. Detection of compounds or products of the bacteria

 PCR-based genetic tests

Detection is based on multiplication not of whole bacilli, as in culture, but of
their genetic material, chromosomal DNA or ribosomal RNA. Provided all
ingredients are present in the reaction tube, this will only take place when the
target genetic sequences to which the added primers can bind are found in the
sample. Specificity of the test will thus depend on the use of correct primers,
using sequences typical for MTB or MTB complex. In principle, from one
target sequence, of one bacillus, the reaction can produce millions of copies and
thus yield a positive result.
In practice, however, sensitivity is of the same order as that of culture or about
100 bacilli, due to the limited amount of sample that can be used and sometimes
also due to the interference of reaction-inhibiting substances. Specificity of
PCR is probably also similar to that of culture, but this needs further evaluation.
Extremely careful technique is needed. Carry-over of DNA is even more likely
to happen than that of whole bacteria so that early studies found up to 50% of
positive results to be false. This places high demands on skills of the
technician, as well as requiring special equipment and infrastructure (i.e.
separate rooms).
The main advantage of PCR-based techniques is their speed; in principle, only
1-2 days are needed. This is true for diagnosis of TB, and even more so for
applications such as diagnosis of drug resistance (mainly rifampicin) and
species identification using probes.
The main disadvantage of PCR-based tests is their extremely high cost,
especially when more convenient and more sensitive commercial test kits are
available. Even in rich countries this has restricted their use, for instance, to
determining the species present in smear-positive disease (FDA approval). This
restricted use may also reduce the speed of the results, since it may not be
possible to schedule PCR-runs daily.
For resource-poor countries, detection of TB using PCR seems unrealistic.

 Chromatography
Various techniques have been described using high-performance liquid
chromatography (HPLC) or gas-liquid chromatography (GLC). Detection
 targets substances from the cell-wall such as mycolic acids, or metabolic
products such as tuberculostearic acid. Results have been extremely good in
terms of sensitivity and specificity as well as speed.
Nevertheless, these techniques seem to be used rarely in routine practice. The
main reason for this may be the expense of the equipment, which has no other
applications in the bacteriology laboratory.

 Antigen detection
See further, serology.

3. Serology

Antigen detection, antibody detection, detection of immune complexes or even

immune-detection of complete bacterial cells, all have been described with
many variations. The simplicity, ease and speed of these methods are
attractive. A really good immunologic test could be applied by almost anybody
and almost anywhere, and yield a result while the patient is waiting. For this
reason, the search for such a test has been continuous since the time of Koch,
with a re-intensification because of new discoveries such as monoclonal
antibodies or recombinant peptides and perhaps now the deciphering of the
MTB genome. New tests are continuously being introduced by scientists as well
as commercial companies, always with big promises. So far, all of them have
been disappointing.
The main problems seem to be that the antibody response to various TB
antigens is highly variable between patients; it is very difficult to find a highly
specific antigen as well. This results in problems of sensitivity as well as
specificity, especially with easy-to-use formats such as latex-agglutination or
immune-chromatographic (dot) tests. Typically, sensitivity is comparable to
that of AFB-microscopy, and clearly better also in smear-positive cases. At the
same time, however, specificity remains well under that of microscopy. At best
it reaches about 95%. While this may seem high, it will lead to about 50% of
false positives in all but high-prevalence regions. Better results are obtained
with more complicated techniques, such as sandwich ELISA, but they are not
useful in routine practice. On the other hand, far poorer results seem to be
obtained in HIV positives. For instance a field evaluation of MYCODOT test
in Tanzania has shown a sensitivity of only 26% in smear-positives, and 7% in
culture- or X-Ray-proven cases, for a specificity of 84%. The table below
shows results from a panel evaluation of various commercialized serological
tests. With this excellent panel composition, mimicking what can be expected
in routine work as to proportions of pathologies to be differentiated, not a single
test scored high enough to be useful.

To conclude, we do not yet have a serological test which is good enough, and
especially not to detect the cases where microscopy remains deficient (HIV
positive, children, EPTB). Considering experiences hitherto, claims in this
regard by the manufacturers should be considered as publicity until field
evaluation has proven their true value.