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. + Interested readers could contact the author for a comprehensive list of books/articles on the subject. 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 10 3 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. . 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. 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. 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