LABORATORY DIAGNOSIS OF PULMONARY TUBERCULOSIS : CONVENTIONAL AND NEWER APPROACHES+ pdf

MICROSCOPY The diagnosis of pulmonary tuberculosis can be made by the detection of acid fast bacilli by direct microscopy, using carbol fuchsin stain and/or fluorochrome stain.. CULTURE

CONTINUING MEDICAL EDUCATION Ind J Tub., 1996,43,107

LABORATORY DIAGNOSIS OF PULMONARY

TUBERCULOSIS : CONVENTIONAL AND NEWER APPROACHES+

N.K Jain*

Inspite of the discovery of the causative

agent more than a century back, the bacteriological

diagnosis of tuberculosis has been a major hurdle

in the treatment and control of the disease Due

to its global prevalence, the methods available

for diagnosis are under rapid development Briefly

reviewed, below are the present and the possible

future laboratory tools for die diagnosis of

pulmonary tubercolosis

MICROSCOPY

The diagnosis of pulmonary tuberculosis

can be made by the detection of acid fast bacilli

by direct microscopy, using carbol fuchsin stain

and/or fluorochrome stain Microscopy is a rapid

method but lacks sufficient sensitivity and does

not distinguish between different species of

mycobacteria The sensitivity of microscopy is

often not more than 25-40% as compared to

culture, but under ideal conditions, it is possible

to achieve a rate of 60-70% The average rate

obtained in India is 12-15%, but microscopy is

still the mainstay of the tuberculosis control

programme of the developing countries, including

India The fluorochrome stain technique is better

then carbol fuchsin stain method, but is very

expensive and less specific, and is not suitable

for adoption as a routine method in our country

CULTURE

Culture is considered as the reference method

for the detection of tubercle bacilli, but

mycobacterial culture is laborious, expensive and

slow It is worth noting, however, that not more

than 50% of clinically diagnosed patients are

confirmed by culture Traditional culture on

Lowenstein Jensen egg medium takes 2-6 weeks

The introduction of other media for culture of

tubercle bacilli, such as 7H10 and 7H11 agar,

ave resulted in a faster & higher rate of

h

I

detection These media an commonly used in developed countries and are costly The radiometric respirometry technique (BACTEC) is more sensitive and can drastically reduce the time needed for detection, the median time being around 4 weeks both for detection and sensitivity testing This technique is very commonly used in the United States, but due to its limitaiions in terms of cost, specificity and biohazards, it is not suitable for developing countries The other rapid culture methods are biphasic broth agar system (Septi- Chek AFB, Becton-Dickinson) and slide culture method The slide culture method seems very good in principle, as it may help reduce the time needed for culture and sensitivity, but it needs standardization before it can be accepted as a routine method

SEROLOGICAL TESTS

The tubercle bacillus, like all other bacteria,

is rich in antigens that stimulate antibody production Several assays have been developed

in the last one century to detect specific antibody response in suspected patiets These assays use either whole bacteria or fragments of AFB, culture filtrates, partially purified antigens and purified antigens both by chromatography and by recombinant DNA technology Various techniques are used to carry out the tests, such as ELISA, radio-immuno-assay and immunoblot Moreover, instead of looking for specific antibodies, attempts have been made to detect antigens in clinical samples using specific polyclonal and monoclonal antibodies There are several inherent difficulties

in hunting for antibodies to diagnose tuberculosis Crude antigen preparations which are likely to share epitopes with most, if not all, wild strains

of M tuberculosis are also likely to share some

epitopes with non-pathogenic environmental mycobacteria Purified antigens, on the other hand, may not be expressed by every patient

* Bacteriologist, New Delhi Tuberculosis Centre, J.L Nehiu Marg, New Delhi - 110 002.

108 N.K JAIN

infected with tubercle bacilli Antibody responses

may persist for years particularly if one accepts

the notion of a dormant population of bacilli that

occasionally surface to reproduce before becoming

dormant again In high prevalence areas, it may

be difficult to distinguish current from old disease

But, to date, no assay is acceptable for the

diagnosis of tuberculosis

The tuberculin test is also used by many

physicians for the purpose of diagnosis It is not

a test for diagnosis as it tells only whether or

not a person is infected with M.tuberculosis, but

cannot differentiate between infection and disease

This test is of no use at all in countries with

endemic tuberculosis where a large part of the

population is already infected with the bacilli It

may only be of use in children where other tests

are not of much help

recent years, the diagnosis of tuberculosis

has ueen facilitated by the detection of

mycobacterial cell wall components

(Tuberculostearic acid or TSA) and increased

production of host enzymes (Adenosine Deaminase)

directly in clinical samples TSA is present not

only in M.tuberculosis, but also in other

mycobacteria as well as other microorganisms

which constitute the normal microflora in various

body sites Thus, this method is not suitable for

diagnosis The sensitivity of ADA test is good

but specificity is low Consequently, this test is

not very popular

DNA PROBES

Recent developments in the field of molecular

genetics of mycobacteria have made it possible

to identify particular sequences of DNA that are

specific for individual mycobacterial species

These unique DNA sequences can be detected

using labelled oligonucleotides that are exactly

complementary to the nucleotide sequence in the

mycobacterial genomic DNA Such DNA probes

can identify genus with high specificity, or

species specific bacterial DNA sequences Although

such probes have been shown to be highly

sensitive and specific, when used in research

laboratories, these lose specificity and sensitivity

when used directly in clinical samples

PCR

Currently, much attention is being focussed

on the use of polymerase chain reaction (PCR) The principle of the PCR technique is based on the amplification of a given DNA sequence to

a large number of copies that can be identified

by separation on gel electrophoresis and, subsequently, either with or without probing with

a labelled oligonucleotide specific for the amplified DNA fragment During the last decade, several such unique sequences have been reported

for the M.tuberculosis complex The usefulness

of PCR for detection of tubercle bacilli in clinical specimens has been confirmed in several recent studies, with sensitivities and specificities ranging from 60% to 100% Contamination is a problem that all PCR laboratories must face, since the product of the reaction contains millions of suitable templates that can be carried back to the next assay on finger tips, pipettes, clothing or in aerosols With mycobacteria there

is the additional difficulty of extracting DNA from within the cells and this is one of the important limiting factors in determining the

sensitivity of PCR assays for M.tuberculosis in

clinical material There have been many reports

in recent years and several groups have published encouraging results involving considerable number

of samples, but routine mycobacteriology laboratories are not yet convinced of its practical utility The results of a recent study organised

by the WHO (Noordhoek et al, 1993) must be

an eye opener for those of us who are carried away by certain reports coming from research publications Batches of 200 samples containing

varying numbers of M.bovis BCG suspended in

water, saliva or sputum were sent, coded, to seven laboratories that had established PCR

assays for M.tuberculosis One of these was

unable to provide results within 6 months Three had results with specificities of less then 80% and the three laboratories, in which false positives were rare, only picked up 60% of samples with

103 organisms in 0.2 ml, just a little improvement

on a good microscopy service At present, there are no commercial PCR kits that have been evaluated and licensed by the Food and Drug Administration (U.S.A) for detection of

M.tuberculosis in clinical laboratories.

LABORATORY DIAGNOSIS OF PULMONARY TUBERCULOSIS 109

RFLP

Molecular genetic methods are also useful

in studying the epidemiology of tuberculosis

Recent outbreaks of multiple drug resistant

tuberculosis _have been traced by DNA finger

printing of tubercle bacilli isolates, using

restriction fragment length polymorphism (RFLP)

The principle of the method is to extract the

mycobacterial DNA from cultured organisms,

digest it with chosen DNA cleaving restrictive

enzyme(s), separate the DNA fragments produced

by gel electrophoresis, whereby certain repetitively

occurring DNA sequences (insertion sequences)

are identified by specific probes This results in

a fingerprint highly specific for each individual

mycobacteral strain This molecular genetic

identification on the sub-species level now makes

it possible to trace the spread of specific strains

in the community This technique may, in the

near future, drastically change the morphology of

tuberculosis laboratories, especially in

industrialized countries But what about developing

countries, which have 2/3rds of the world’s

population and almost 90% of patients? Do we

foresee that the fruits of these newer technologies

can help the developing countries control the

problem of tuberculosis in the near future? I

would like to suggest that we should stick to the

original established tools for the diagnosis of

Tuberculosis and try to improve upon our own

deficiencies for making the best use of the old

technique of microscopy instead of criticizing the

inherent deficiencies of this technique If we can

do so, this old but time tested technique can

match the newer technologies as far as tuber-

culosis control is concerned The saying that

OLD is GOLD stands true still Lipsky, in a

review of factors affecting the clinical’ value of

microscopy for acid fast bacilli, concludes that

when the results of all specimens from each

patient are considered in total, the acid fast smear

has a predictive value of >96% and remains one

of the most rapidly performed tests in the

detection of pulmonary tuberculosis All other

techniques need to be compared critically with

microscopy and their diagnostic role assessed

The traditional model of history, examination and

sputum microscopy has proved itself capable of

detecting the majority of infectious cases and, in

conjuction with adequate case-finding and

treatment, remains the backbone of tuberculosis control programmes

DRUG SUSCEPTIBILITY

The role of drug sensitivity testing should not be underestimated in the treatment of difficult cases of tuberculosis This is another very important area where the laboratory plays an important role Anti-tuberculosis therapy depends on the susceptibility of the tubercle bacilli The newer regimens include four drugs because of increase

in initial drug resistance levels in the high prevalence countries

Drug resistance may be defined as the ability of strains of tubercle bacilli to survive and grow despite exposure to concentrations of the drug that inhibit or kill the parental bacilli, and to transfer this characteristic to its progeny

It has also been defined as a decrease in sensitivity to a drug of sufficient degree to make it reasonably certain that the strain concerned is different from a sample of tubercle bacilli that have never come into contact with the drug At present, the only known

mechanism for M tuberculosis’s acquisition of

drug resistance is spontaneous, mutation To date, there is no clear evidence indicating that tubercle bacilli acquire resistance through resistance transfer factors or other genetic mechanisms

Untreated tuberculosis patients infected with drug-resistant bacilli are referred to as having

‘primary’ drug resistance, to distinguish them from those who had drug-sensitive organisms orginally and later developed resistance because

of inadequate or inappropriate chemotherepy The latter are generally referred to as having

‘acquired’ drug resistance A better term for

‘primary’ drug resistance might be ‘initial’ drug resistance, because what is reported in the literature

is usually a mixture of primary drug resistance with an unknown degree of acquired drug resistance Recently, another term - multi-drug resistant tuberculosis (MDR-TB) - has been added and is defined as “drug resistance against two most potent drugs i.e., Isoniazid and Rifampicin with or without resistance against any other drugs”

110 N.K JAIN

Three widely used methods for testing

sensitivity are the proportion method, the resistance

ratio method amd the absolute concentration

method Resistance ratio method is most

commonly used in developing countries, whereas

the proportion method is common in industrialized

countries The more rapid method commonly

used in U.S.A is the radiometric system, the

BACTEC, and is a modification of conventional

proportion method The test uses Middlebrook

7H12 broth with a radio-labelled fatty acid

substrate The amount of growth, indicated by

changes in the growth index in the media with

known drug concentrations as compared to that

in the control bottle, has been correlated to the

presence or absence of resistance in 1% of the

inoculum Thus, if an isolate grows beyond a

specific growth index compared with the control,

it is considered resistant to the specific agent

This method of testing has a very rapid turnaround

time, with results for sensitivity usually available

in less than 7 days This method is very costly

both in establishment and recurring expenses,

besides being hazardous It may not be possible

to use this as routine method in the developing

countries in the near future The resistance ratio

or proportion method uses the routine LJ medium

and takes 4 weeks The use of 7H10 or 7H11

medium instead of LJ for drug susceptibility can

reduce the time by one week The use of slide

culture method can reduce the time considerably,

but needs standardization before being used as

a routine method

Innovative methods for rapid drug testing

are currently being developed In one such

method, mycobacteria are infected with specific

reporter phage expressing the firefly luciferase

gene Light production is dependent on phage

infection, expression of the luciferase gene, and

the level of cellular ATP Signals can be detected

within hours of infection of virulent M.tuberculosis

isolates with reporter phage When organisms are

exposed to drugs to which they are susceptible,

the-light is extinguished unlike strains resistant

to that drug The result may be available within

two days The method is still in its developmental stage

Another approach for the rapid determination

of drug susceptibilities is based on potential difference between the genetic material of a drug resistant strain and that of a drug susceptible strain This difference may be applied for Isoniazid resistance detection by detecting the presence or

absence of Kat G and INHA genes associated with Isoniazid resistance to M.tuberculosis There

is also a good possibility of detecting Rifampicin resistance by a PCR based assay that detects difference between Rifampicin susceptible and

Rifampicin resistant strains The rpoB gene which

encodes the RNA polymerase subunit b, has been cloned and ‘mutations in this gene have been identified in rifampicin resistant strains but not

in rifampicin sensitive strains The rapid screening

test for rpoB gene mutations has been successfully

carried out by using PCR single-strand confirmation polymorphism (SSCP) technique Similarly,

resistance to Streptomycin in M.tuberculosis is due to mutations in rpsL gene which encodes the ribosomal protein S12 and rrs gene which encodes

16S rRNA With PCR-SSCP technique, the

mutations in rpsl and rrs genes have been detected in clinical isolates of M.tuberculosis

resistant to Streptomycin Similarly, different PCR-SSCR patterns of mutations in codons of gyrA have been found to be associated with resistance to Ciprofloxacin This novel technique, PCR-SSCR, can detect drug resistance in 48-72 hours However, rapid identification on molecular genetic principles may be too intricate to be practical, at least in the developing countries The diagnosis of tuberculosis and antimycobacterial susceptibility testing are important problems confronting laboratory, physicians and scientists However, until new technologies and techniques are well established,

we should make good use of old and time tested techniques to their maximum efficacies to treat, manage and control tuberculosis as well as expand the boundaries of our knowledge