It is necessary to make a close observation on contamination rate. If the contamination rate is over 5% it indicates improper decontamination of specimens and, if less than 2%, may indicate too harsh pretreatment.
C. Indentification
As far as the identification tests are concerned, a simple and rapid differentiation of M. tuberculosis from other mycobacteria (MOTT) is most important. This can be done easily by simple biochemical tests such as niacin test, nitrate reduction test and 68 ℃-labile catalase test, if the growth is affluent, in addition to the growth characteristics, namely delayed appearance of rough, hard-textured, ivory-colored colonies showing serpentine cord under microscope. If the growth is scant and so not enough for biochemical tests, then it is recommended to subculture them not only on drug-free medium but also on the medium containing 500μg/ml of p-nitrobenzoic acid (PNB). if M. bovis infection is suspected, the medium containing 5μg/ml of thiophene-2-carboxylic acid hydrazide (TCH) should be included. Almost all of MOTT grow on both media, whereas nearly all of M. tuberculosis including M. bovis do not grow on PNB medium and the latter species does not grow on both media. Some asian variant of M. tuberculosis do not grow on TCH medium, thus biochemical tests may be necessary for confirmation of M. bovis.
D. Drug susceptibility test
Drug susceptibility test is very complicated, time-consuming, and expensive procedure that requires highly skilled technique and must be performed according to the well-standardized technique under strict quality control, otherwise it will not only waste resources but mislead clinicians to improper treatment of patients or the authority in planning of NTP.
Drug susceptibility testing is so complex and laborious that reliable results are hardly obtainable on a consistent basis unless the tests are performed according to the well standardized technique without deviation and under strict quality control. In this regards, it is desirable to perform DST only at the central or national reference laboratory.
1. Use of drug susceptibility tests
(a) It is generally accepted that drug susceptibility tests would provide the best possible guidance to clinician in the retreatment of patients who failed initial treatment. But DST of course is not necessary for initial treatment.
(b)DST may provide useful epidemiological information, for example, prevalence of drug resistance can be used to evaluate the past and current treatment programme and may help plan the effective treatment programme such as type of chemotherapy that could be used for patients self-presenting at HC for the first time.
2. DST methods
If sputum specimen contain numerous bacilli, DST can be performed by direct method, which has some merits, for instance, inoculum is more representative of those present in the lesions and it takes much shorter time to get result than indirect test with an average gain of 3-4 weeks. However it is not easy to standardize the inoculum size because viability of organisms in the specimen varies according to the duration of storage and decontamination procedure. Indirect test is inevitable in case of smear negative culture positive, contamination is present in the direct test, growth on drug free medium is scant and so unable to issue reliable report, and the cultures are submitted from other institutes. The cultures grown on L-J medium can be stored at 4 ℃ without change of drug susceptibility pattern. If stored at -20 ℃ or -70 ℃ after suspending the organisms in skim milk, they can be stored for many years.
There are basically three different techniques, called the absolute concentration method, the resistance ratio method, and the proportion method. Of these the proportion method is most widely used in many laboratories. This method was introduced by Middlebrook & Cohn. Inoculum preparation is same as in (1) and 1mg/ml bacterial suspension is diluted 10-3 and 10-5 mg/ml by serial 10 fold dilution. Then 0.1ml of dilution is planted onto each slope of drug-free or drug-containing medium. The result is read on the 28th day of incubation at 37 ℃, if the growth on drug free medium is poor, a second reading on the 42nd day. Report proportion of colonies grown at the critical concentration of drug. In general, 1% is used as a critical proportion for all drugs.
3. Criteria of resistance
Whichever method is used, it is not easy to determine the rational criteria of drug resistance. The critical concentrations and/or critical proportions, by which the susceptible strains are separated from resistant strains, should be determined by analysis of MICs or resistant proportions of probably sensitive clinical isolates from never treated
