Comparative efficacies of three acid-fast staining techniques under field conditions for Mycobacterium tuberculosis in the Indian context

摘要

Tuberculosis is a most important human disease in its global prevalence. It is caused by tubercle bacilli. Among the various methods for diagnosis of pulmonary tuberculosis, bacteriological methods are more important. Standard Ziehl-Neelsen method of staining is most reliable for this. But due to some operational disadvantages in the Ziehl-Neelsen method, many other methods were also discovered. In this research work two such techniques that is Modified Ziehl-Neelsen method where a boiling water bath is used as a source of heating and cold staining method where heating is completely eliminated and instead of which duration of exposure to carbol-fuchsin stain prolonged to 10 minutes were compared with conventional Ziehl-Neelsen method. For these 145 sputum samples having the symptoms of tuberculosis were collected and each of these samples were stained by three above mentioned methods. The results were analyzed by Mc Nemar’s chi-square test and kappa statistics. It was observed that Cold-staining method is more appropriate than that of the Modified Ziehl-Neelsen method. Additionally due to some advantages in cold staining method it is more reliable and safer than that of the standard Ziehl-Neelsen method in field conditions. Introduction Tuberculosis is a communicable disease due to infection by tubercle bacilli. Every year approximately 8.8 million new cases of tuberculosis are detected [1]. About 20% of the world’s population of tuberculosis patients resides in India [2]. According to WHO expert committee, a case of tuberculosis refers to a person with ‘bacteriologically confirmed disease’ that is a person who expectorates tubercle bacilli in his sputum [3]. As per as report given by WHO, there were 8.8 million new cases of tuberculosis globally in 2003 with an expected 1% increase annually and 1.7 million deaths worldwide [4,5].This disease is also associated with human immunodeficiency virus infection (HIV) [5]. About 9% of tuberculosis infected patients are HIV positive. The rate of infection and risk is higher in Asian countries including India with a 35 per cent of all cases of worldwide alone in India and China[5] Correct and rapid diagnosis of the disease is very important to control the growing epidemics [4, 6]. Laboratory diagnosis of pulmonary tuberculosis is mainly dependent on the demonstration of acid-fast bacilli in sputum by direct microscopy or by culture methods [7]. Smear microscopy being simpler, quicker and cheaper is widely used for detecting tubercle bacilli [8] though it lacks sufficient sensitivity [7]. Culture of mycobacterium is also beneficial for diagnosis of this disease, but the organism is slow growing and the method is laborious as well as expensive. There are some other good methods for diagnosis such as BACTEC radiometric system which need 4 weeks for detection and sensitivity testing and other rapid culture methods including Septi-Chek AFB, Becton-Dickinson and slide culture method [7]. Recently amplification of mycobacterial DNA using PCR is in practice in developed countries, but not in developing countries [6]. The methods except direct microscopy are expensive, biohazardous and need special set up for working, this is for that reasons these methods are not universally available [7]. So, the direct microscopy technique is more reliable if we can improve their deficiencies [7]. Ziehl-Neelson’s staining is commonly used for detecting tubercle bacilli. This technique requires heating of the smear, while staining heating is usually done either by using spirit lamps or by Bunsen burners. Further, in this technique a high degree of precision is required i.e. overheating may char the smear and under heating may result in pale staining of the bacilli. Number of modifications has been made in this technique to avoid the heating or use of flame [9, 10, 11, 12, 13, 14, 15, 16, 17]. In modified ZN method (MZN) instead of heating directly in the flame the slides are placed on water bath and heated by