Establishment And Clinical Application Of Cerebrospinal Fluid Modified Acid Fast.

Objective: Tuberculous meningitis(TBM) is an infectious disease of central nervous system caused by Mycobacterium tuberculosis, which is one of the most serious types of tuberculosis. Without timely treatment, it is destructive and has a very poor prognosis, so early diagnosis and prompt treatment are critical to the reduction of morbidity and mortality. TBM diagnosis is the most difficult, for it tends to be misdiagnosed as viral meningitis, bacterial meningitis or carcinomatous meningitis. It is the golden standard for TBM diagnosis to identify or culture M. tuberculosis [1]. However, there are fewer cerebrospinal fluid(CSF) specimens, while the traditional smear acid-fast staining and Mycobacterial culture require a lot of CSF specimens with low M. tuberculosis detection rate, which has been reported no more than 10% [2-3]. Traditional Mycobacterial culture method Lowenstein-Jensen takes a long time, about 4-6 weeks, to detect the growth of M. tuberculosis [4], and even the existing BD Fluid Thioglycollate Medium also takes 2-3 weeks [21], which impact the disease diagnosis and follow-up treatment. Serological methods are convenient but lack of sensitivity and specificity, often used for assistant diagnosis in clinical practice [5]. In recent years, the development of DNA technology and other fast culture technologies has reduced the detection time of M. tuberculosis, but due to the high price, they are not suitable for widespread use in clinical practice. This study aims to explore a fast, efficient and economical clinical detection method to promote the extensive survey of TBM. The acid-fast staining is the best diagnostic method of M. tuberculosis infection, but it has a low detection rate for TBM diagnosis. To this end, a parallel comparison was made between the modified acid-fast staining based on traditional acid-fast staining and a variety of TBM diagnostic methods by the clinical laboratory(including condensing acid-fast staining, traditional acid-fast staining, CSF ADA, FQ-PCR, etc.) and analyzed mutual relations in the disease development in order to understand the detection rate of modified acid-fast staining in TBM diagnosis, which highlight its fast, efficient and easy operation and provide a theoretical basis for the screening of TBMMethodology:1 A total of 216 CSF specimens were collected from 64 suspected TBM patients in Internal Medicine-Tuberculosis and Neurology, Hebei Chest Hospital, from January 2014 to October 2014(not less than 3 specimens were collected from each patient and the time interval was not less than one week), and all patients hospitalized in January 2014.2 Index detection:216 specimens be detected by the modified acid-fastaining from 64 suspected TBM patients, in the meantime to be tested by CSF RT, CSF condensing acid-fast staining, CSF smear staining, CSF ink staining, CSF biochemical test(including glucose measurement,Cl ion measurement, protein measurement and ADA measurement) and CSF cytology(on the first or second day of hospitalization, Mycobacterial culture and common bacteria culture of CSF were carried out for 64 specimens,and FQ-PCR was carried out for 27 patients).3 index analysis: the modified acid fast stain results compared with other conventional test results, comparative positive rate difference, integrated with the clinical indexes, the final diagnosis for tuberculous meningitis4 Statistical analysis: using chi-square test comparing detection rate with and without difference. Inspection level of α= 0.05.Results:1 After the experiment, read the clinical data:31 illness took a turn for the better without antituberculosis therapy, Cryptococcus infection confirmed in 3 patients,There were 4 meningeal carcinomatosis which doubt was brain metastases from lung cancer,24 cases iwere viral meningitis or bacterial meningiti. So this subject set up Tuberculous meningitis positive group with 33 cases and Tuberculous meningitis negative groups with 31 specimens?2 M. tuberculosis morphological observation: under an optical microscope, they had a light blue background and a thin red rod shape, but some of them were slightly curved. Piles of palisade-shaped M. tuberculosis were occasionally seen. Most of them were extracellular bacteria, but intracellular bacteria were occasionally seen.3 The 27 specimens of TBM group were detected DNA of M. tuberculosis by CSF FQ-PCR, 23 of them were positive. 33 patients were detected by Modified acid-fast staining 29 of them were positive. The positive rates of the two methods were compared P>0.05, the two methods were not statistically different.4 Comparison between modified acid-fast staining and traditional acid-fast staining: 65 specimens tested by the modified acid-fast staining were positive in the 107 specimens of the 33 patients and the positive rate was 60.75%; 6 specimens tested by the traditional acid-fast staining were positive in the 107 specimens of the 33 patients and the positive rate was 5.61%. Therefore, the modified acid-fast staining had a higher positive rate of Mycobacterium tuberculosis in CSF than the traditional acid-fast staining,P<0.005 and the two methods were statistically different.5 The relationship between the accounts of cell in CSF and the positivity of modified acid-fast stain: According to the cell accounts, the CSF specimens depart into two groups, 39 of 65 CSF specimens are positive in less than 100 group, the detectable rate is 60%. 26 of 42 CSF specimens are positive in more than 100 group, the detectable rate is 61.9%. P>0.05 There is no significant difference between the two group6 Comparison between Mycobacterial culture and modified acid-fast staining: for modified acid-fast staining, there were 65 positive specimens in the 107 specimens in the patient group and the positive rate was 60.75%; for Mycobacterial culture, there were only 3 positive specimens and the positive rate was 2.8%. Therefore, the detection rate of modified acid-fast staining was higher than Mycobacterial culture, P<0.005 the two methods were statistically different.7 Relationship between modified acid-fast staining and CSF cytology: for CSF cytology analysis of these 64 specimens of patients with suspected intracranial infection, hypersegmented neutrophils accounted for more than 60%, and with the treatment, segmented neutrophils showed a progressive decline, while activated monocytes and lymphocytes increased. For these 64 specimens of suspected patients, multiple parallel analyses of CSF cytology and modified acid-fast staining were carried out, 27 specimens with a large proportion segmented neutrophils were found to have acid-fast bacilli, and the number of cells showed a progressive decrease with the continuation of the treatment time. Therefore, for the specimen with more segmented neutrophils in CSF cytology, the detection rate of M. tuberculosis was significantly higher than that with a high proportion of lymphocytes in CSF cytology, P<0.05, they were statistically significant.8 Comparison between modified acid-fast staining and ADA in CSF biochemical test: ADA activity increased significantly in the early TBM, and ADA and modified acid-fast staining were respectively carried out for CSF of 216 specimens. In the parallel contrast, ADA activity significantly increased in 78 specimens, and 59 specimens of modified acid-fast staining were positive. In the remaining 138 specimens of CSF, ADA activity was relatively low, and there were 6 specimens with acid-fast bacilli detected. According to the statistical analysis P<0.05,they were statistically significant.9 Comparison between modified acid-fast staining and condensing method acid-fast staining: 29 specimens tested by the modified acid-fast staining were positive in the 33 specimens and the positive rate was 87.9%; 6 specimens tested by the condensing method acid-fast staining were positive in the 33 specimens and the positive rate was 18.2%. Therefore, the modified acid-fast staining had a higher positive rate of Mycobacterium tuberculosis in CSF than the condensing method acid-fast staining, P<0.05,the two methods were statistically different?10 The relationship between modified Z-N stain and before and after treatment:33 specimens CSF were detected by modified Z-N stain before antituberculosis treatment,74 specimens CSF after antituberculosis treatment,Positive rates of the two groups were compared,P<0.05, the difference was statistically significant.Conclusions:1 Compared to traditional acid-fast staining, condensing acid-fast staining and Mycobacterial culture, modified acid-fast staining significantly increases the positive rate of acid-fast bacilli and saves the diagnosis time with low false positive rate and simple operation. Therefore, it has a very high value for the rapid and accurate clinical diagnosis of TBM and can be taken as the best screening test for TBM.2 For CSF specimens of the same TBM patient, the change in cell component ratio is significantly correlated to the detection rate of M. tuberculosis. The positive rate of CSF specimens with more segmented neutrophils is significantly higher than those with a higher proportion of other cells. The change in cell component can be used as an index to observe the condition of infectious diseases of nervous system.3 ADA can be used as the early assistant diagnosis of TBM as well as RT to observe the disease and efficacy.4 Compared to FQ PCR, modified acid-fast staining is not significant different in the positive rate of M. tuberculosis in CSF and has a lower cost, so it can be used as a screening test for suspected TBM in clinical practice.5 If the patient is suspected with TBM, the CSF should be collected and tested by modify acid-fast stain before the chemotherapy.