BACKGROUND: We evaluated the effects of changes in laboratory practices on the detection rates and turnaround time in a clinical mycobacteriology laboratory. METHODS: A total of 27,339 specimens from 9,947 patients were tested during the period from September 2000 to March 2004, which could be divided into the following four periods according the culture and identification methods used: Period I, use of 2% Ogawa medium for culture and niacin for identification; period II, introduction of PCR for identification; period III, introduction of BacT/Alert system (bioMerieux, Durham, USA) for culturing sterile body fluids; period IV, use of Bact/Alert system for CSF and the second of repeated sputum specimens from the same patients. During these periods, two technicians (one for the first half and the other for the second half periods) did all mycobacterial tests except PCR. RESULTS: Mean detection rates were 8.0% by auramine stain, 7.9% by nested PCR, and 6.6% by culture. The detection rates by culture for sputum specimens varied 11.4%, 7.7%, 8.1% and 5.9% in each of the four periods; for body fluids, the detection rate of 4.3% during the period III was the highest. The proportion of stain results reported within 24 hours and the identification of culture isolates within 21 days changed from 80.9% to 72.4% and from 2.3% to 30.8%, respectively. With an introduction of PCR for identification in the period II the time required for the identification of cultures decreased dramatically from 26.5 days to 4.8 days. The TAT of a direct detection by nested PCR changed from 7.2 days to 5.1 days. CONCLUSIONS: New tests should be introduced in the clinical mycobacteriology laboratory. But the cost and workload of the tests should be taken into consideration to make the laboratory service more efficient.
Benzophenoneidum ; Body Fluids ; Humans ; Niacin ; Polymerase Chain Reaction ; Sputum ; Tuberculosis

BACKGROUND: Currently, many laboratories have selected several different methods for the detection of M. tuberculosis in the sputum. To select efficient method for clinical laboratories among the various methods, we compared the results of several methods. METHODS: Total 72 sputums were examined by the six combinations of stain methods. The samples were constructed as follows on the result of direct smear ZN stain; negatives (26), traces (3), 1+(9), 2+(12), 3+(12) and 4+(10). The true positives were determined after close evaluation of the clinical, radiological and other laboratory findings. RESULTS: The sensitivities and specificities of each methods were as follows; direct smear ZN stain were 83.6% and 100%, direct smear Auramine stain were 90.9% and 100%, centrifugation ZN stain were 94.6% and 100%, centrifugation Auramine stain were 98.2% and 94.1%, cytocentrifugation ZN stain were 96.4% and 100%, cytocentrifugation Auramine stain were 100% and 64.7%, nested PCR were 80% and 94.1% and culture were 67.3% and 100% respectively. CONCLUSIONS: Concentration method by centrifugation is suitable for routine laboratory if enough centrifugal force were engaged. Auramine stain is more suitable staining method than ZN stain in direct smear but not in concentrated smear because it has the potency of false positivity. The PCR assay is thought to be not only a fast, sensitive method but also a specific method for the direct detection of M. tuberculosis in the sputum. The culture method using Ogawa media is specific but not sensitive.
Benzophenoneidum ; Centrifugation ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymerase Chain Reaction* ; Sputum* ; Tuberculosis

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the mycobacteria. However, this method has several issues of false negative results, and hence a comparative experiment of the Ziehl-Neelson's AFB staining and the fluorescence staining method was done to remedy this problem. As the fluorescence staining method brightly highlights the AFB in a dark field, and also as it is observed with the lower power objective, it is a method that can better the observation and shorten the time of observation as well. The fluorescence staining method that was used in this experiment did a comparative analysis of the Auramine O-Rhodamine B and the Acridine Orange. The results showed that although the Auramine O-Rhodamine B allows easier observation of the AFB with a high fluorescence expression rate for the multibacillary leprosy sample, the darkness on the periphery makes it hard to observe anything else, while also making it hard to observe the cell changes and paucibacillary leprosy of the AFB. However, the Acridine Orange staining method highlights the cells in dark green and changes the color of the AFB from bright red to orange making it easier to observe bacilli. The results of the study show that the Acridine Orange method is superior to the Auramine O-Rhodamine B method in detecting acid fast bacilli in specimen.
Acridine Orange ; Benzophenoneidum ; Citrus sinensis ; Darkness ; Fluorescence ; Leprosy, Multibacillary ; Leprosy, Paucibacillary ; Mycobacterium ; Mycobacterium leprae

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the diagnosis of leprosy. However, the fluorescent stain performs better and allows the detection of more positive smears. The limitation for its widespread use has been the high cost for fluorescent microscopes. Novel light-emitting diodes (LED) are inexpensive solutions for fluorescent microscopes, and thus fluorescent stain may be a cost-effective step to improve the diagnosis of leprosy in resource-poor countries. And the comparison of auramine and acridine orange for staining of acid-fast bacteria was showed significantly more acid-fast rods after using acridine orange and the number of "false positive" results was somewhat higher on auramine staining. So acridine orange offers a good alternative to auramine which is considered carcinogenic. This study evaluated the comparison of the Ziehl-Neelson's AFB stain and the acridine orange stain in the skin smear based on PCR. As PCR results were taken as gold standard, results of the study revealed that the sensitivity of Ziehl-Neelson's AFB stain was 50% and that of acridine orange stain was 92.2%. This study confirmed that the fluorescence stain method is more sensitive than the Ziehl-Neelsen's staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.
Acridine Orange* ; Bacteria ; Benzophenoneidum ; Diagnosis ; Fluorescence ; Humans ; Laboratory Personnel ; Leprosy ; Microscopy, Fluorescence ; Mycobacterium leprae* ; Mycobacterium* ; Polymerase Chain Reaction ; Skin