in 30% after 4 weeks, and also protected from contamination significantly. This explains why sputum specimens should be stored at low temperature. If refrigeration of sputum is not possible and ambient temperature is over 25 ℃ as is often the case in many developing countries, then you may need to use some measure to protect sputum from contamination and to preserve viability of the organisms. The following preservative measures can be used under the routine condition if available.
(1) The fresh sputum is mixed with an equal volume of cetylpyridium chloride (CPC. 1.0%) or cetylpyridium bromide (CPB. 0.6%). Tubercle bacilli can survive for at least two weeks without a great loss of viable counts.
(2) The fresh specimen is mixed with anhydrous sodium carbonate in the proportion 2ml of specimen to 50mg of the reagent.
(3) If the delay before culture examination is to be less than 24 hours, the specimens may be mixed with an equal volume of 23% trisodium phosphate.
Tubercle bacilli can survive in 0.5% CPC for up to three weeks without great loss of viable counts. However CPC(B) easily recrystallize at the low temperature (＜21 ℃) and will inhibit the growth of MTB if inoculated the sediments containing it after centrifugation. Direct sunlight and heat are also harmful to the viability and staining property of tubercle bacilli. For this reason the best efforts should be made to store sputums in a cool and dark place. And it is desirable to collect specimens before drug administration if they are to be cultured.
Some investigators recommend to inoculate specimen onto media at the peripheral laboratory and then to send them to the intermediate or central laboratory in order to overcome contamination and viability loss of the organisms. However, whether this approach is operationally feasible in the developing countries is in question.
?. SELECTION OF LABORATORY PROCEDURES
The clinical diagnosis of TB must be confirmed by bacteriological examination, which is positive by direct smear and/or culture in more than 85% of the patients in technically advanced countries. The proportion of bacteriologically confirmed cases, however, varies with medical practices and thus it is low in some countries, where diagnosis of TB is heavily depended on radiology. If radiologically active but sputum negative cases are put on treatment, over 60% may receive unnecessary treatment (Table 6).
Although there are a variety of laboratory procedures to demonstrate tubercle bacilli in sputum or other clinical specimens, the basic technique in current use have not been changed much since R. Koch and his contemporaries had developed.
However, a variety of new techniques based on molecular biology appeared recently and require clinical evaluation under the various circumstances taking into account of cost-effectiveness. The new techniques include: detection of specific mycobacterial components in clinical specimens, like tuberculostearic acid, using Gas Chromatography-Mass Spectroscopy: nucleic acid amplification methods using polymerase chain reaction technology (PCR); the use of specific DNA-probes; early detection of growth by CO2 production or O2 consumption; and serological methods.
