| Objective: To establishment and evaluate a modified method of the Ziehl-Neelsen stain and determine if it improves detection rate of acid-fast bacilli(AFB) in bronchoalveolar lavage fluid(BALF) specimens.Methods: Bronchoscopy of smear-negative pulmonary tuberculosis(PTB) suspects was conducted to collect BALFs. The formed elements of BALF specimens digested with 4%(weight/volume) sodium hydroxide(Na OH) were collected by using the cytosipin to establish the modified method of the Ziehl-Neelsen stain. BALFs were detected by modified L?wenstein-Jensen(LJ) cultrue, direct smears, concentration smears, and the modified method, respectively. Moreover, the specimens of patients who were cultrue-positive for Mycobacterium tuberculosis(MTB) were tested by Gene Xpert? MTB/RIF. Finally, the efficacy and accuracy of the four methods were compared when culture on LJ medium was considered as the reference standard.Results: A total of 379 patients suspected of having active PTB were enrolled. However, 32 patients were excluded during the study period: 5 were excluded because of acatalepsia, 15 because of missing data, and 12 because of culture contamination. Therefore, 347 patients were included in the final analysis. Of these, 74 were diagnosed with PTB based on BALFs that were culture-positive for MTB by growth on LJ medium. When analyzed by patient, the sensitivity of our modified method was 87.8%(95% confidence interval [CI]: 84.4, 91.2)(n=65/74), which was significantly better than that for direct smears(16.2%; 95% CI: 12.3, 20.0)(n=12/74) or for concentration smears(37.8%; 95% CI: 32.7, 42.9)(n=28/74)(P<0.05 for both comparisons). The specificity was 100%(n=273/273) for direct smears and concentration smears, but 99.6%(n=272/273) for our modified method owing to one AFB-positive, culture-negative case. The positive rate of Gene Xpert? MTB/RIF assay was 90.5%.A total of 106 BALF specimens were collected from these 74 PTB patients. When analyzed by the 106 samples, the number of positive samples detected by culture, direct smears, concentration smears and Gene Xpert? MTB/RIF assay was 81(81/106; 76.4%), 14(14/106; 13.2%), 48(48/106; 34%) and 99(99/106; 93.4%), respectively, while 97 samples(97/106; 91.5%) were detected as positive by our modified method. The positive rares of the modified method and Gene Xpert? MTB/RIF assay was equivalent, which were higher than those of direct smears and concentration smears. The differences were statistically significant(P<0.05 for both comparisons).For quantitative analysis of AFB, 300 fields on each slide from 106 BALF specimens were observed. While the number of AFB-positive fields for direct smears and concentration smears was 34.4 ± 54.0 and 38.2 ± 36.8, respectively, 69.0 ± 67.8(P<0.05 for both comparisons) AFB-positive fields were recorded using the modified method.Conclusion:(1)The modified method of the Ziehl-Neelsen stain is successfully established.(2)The sensitivity of our modified method was significantly higher than that of direct smears or concentration smears.(3) The modified method improved the detection rate of AFB in BALF specimens, and provided an efficient and accurate diagnosis of PTB in smear-negative PTB suspects.BackgroundTuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis. It has higher infection rate and mortality has been the attention of the world’s public health and social problem. China takes about 1/2 of the people infected with Mycobacterium tuberculosis in whole life. Tuberculous pleurisy as the second most common extrapulmonary tuberculosis accounts for approximately 5% of all types of tuberculosis, and 10%~20% of pulmonary tuberculosis. The rate of its incidence is about 1.25%, almost 16 millions of people. The main susceptible population is the adolescent. It is about 73% patients under the age of 39 years, of which 10%-30% of the patients have the manifestation of pleural effusion. If the diagnosis of tuberculous pleurisy is delayed, the pleural thicking, adhesion, and then serious lung function decline and loss of ability to work can be caused.Pleural eff usion is a fr equent clinical problem, and the leading causes of it are tuberculosis and malignant diseases, which have an occurrence of 49.6% and 29.6%.Early diagnosis of tuberculosis is often difficult because the positive rate of the diagnostic standards, such as the Ziehl-Neelsen stain or bacterial culture, is extremely low for pleural effusion. Results of mycobacterial culture requires weeks to be obtained. The PPD is not easy to distinguish with latent tuberculosis infection and BCG vaccination and non-tuberculosis mycobacterial infection. Biopsy of pleura is traumatic, and the operation is time-consuming. Thus, the diagnosis and treatment of tuberculosis can be delayed resulting in increased mortality. Therefore, the current urgent is to develop some efficient methods for the diagnosis of tuberculous pleuritis.In recent years, some researchers modified the Ziehl-Neelsen(ZN) staining method, involving cytospin slides with Triton processing, to improve the detection rate of acid-fast bacilli in cerebrospinal fluid from the 16.7% of conventional ZN staining to 100%. We hypothesize that the use of cytospin slides may increase the sensitivity of AFB microscopy in tuberculous pleural effusion via an exponential increase in the density of bacilli concentrated in a smaller area. Therefore, in this study, we evaluate whether tuberculous pleuritis can be diagnosed using the cytospin slide method.Objective: To establishment and evaluate a modified method of the Ziehl-Neelsen stain and determine if it improves detection rate of acid-fast bacilli(AFB) in tuberculous pleural effusion specimens.Methods: 184 patients diagnosed with tuberculous pleuritis(ADA in pleural effusion >45 U/L) and 43 patientes with tumor from 1 June 2014 to 31 August 2015 at Hospital of Infectious Diseases in Handan and Hebei Chest Hospital were prospectively enrolled in this study. Pleural effusion specimens were collected by using the cytosipin to establish the modified method of the Ziehl-Neelsen stain. The specimens were detected by concentration smears, BACTEC 960 cultrue, Gene Xpert? MTB/RIF assay and the modified method, respectively. Finally, the positive rate, sensitivity and specificity of the four methods were compared.Results:A total of 184 patients with tuberculous pleuritis and 43 patients with tumor were enrolled. However, 5 patients with tuberculous pleuritis were excluded during the study period: 1 was excluded because of missing data, and 4 because of culture contamination. 3 in 43 cases was excluded because of culture contamination, as well.Therefore, 179 patients with tuberculous pleuritis and 40 patients with tumor were included in the final analysis. Of 179 patients, 98 had not been treated, and 81 had. The positive rates of our modified method were 33.67%(33/98) and 25.93%(21/81), respectively, which were higher that those of concentration smears, BACTEC 960 cultrue and Gene Xpert? MTB/RIF assay. The positive rate in the 98 patiens of concentration smears, BACTEC 960 cultrue and Gene Xpert? MTB/RIF assay were 2.04%(2/98) 20.41%(20/98) and 15.31%(15/98), respectivey, and they were 1.23%(1/81), 13.58%(11/81) and 8.64%(7/81), respectively in the 81 cases. The positive rates of modified method were higher that those of other three methods(P<0.05). Whether the patients had been treated or not, the differences of the positive rates of the same mehod had no statistical significance(P>0.05).When the clinical diagnosis was considered as the reference standard, the sensitivities of concentration smears, modified method, BACTEC 960 cultrue and Gene Xpert? MTB/RIF assay were 1.68%(95% CI: 0.00, 3.38), 30.17%(95% CI: 24.09, 36.25), 17.32%(95% CI: 12.31, 22.33) and 12.29%(95% CI: 7.94, 16.64), respectively. And the specificities were 100%(40/40), 100%(40/40), 100%(40/40) and 97.50%(39/40), respectively. The sensitivity of our modified method was higher, and the differences were statistically significant(P<0.05).Moreover, the modified method was used to detect the 179 samples for 3 times, and the detection rates of AFB were 29.05%(52/179), 29.61%(53/179) and 30.17%(54/179), respectively. However, there was no statistical significance among the three times(P>0.05).As to the qualitative estimation of bacterial load, the majority were categorized by Xpert as “very low”(20/22) or “low”(2/22).Conclusion:(1)The modified method of the Ziehl-Neelsen stain is successfully established in tuberculous pleural effusion.(2)The sensitivity of our modified method was significantly higher than that of concentration smears, BACTEC 960 cultrue or Gene Xpert? MTB/RIF assay.(3) The modified method improved the detection rate of AFB in pleural effusion specimens, and provided an efficient and accurate diagnosis of tuberculous pleuritis. |
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