Study On The Value Of The Modified Acid-fast Staining Method In The Diagnosis Of Tuberculous Meningitis

Objective: Tuberculous meningitis (TBM), accounts for5-7%ofsystemic tuberculosis, is the most critical disease in extra pulmonarytuberculosis. As one of the worldwide public health problems, its mortalityand morbidity is very high, rapid diagnosis and early treatment are the keys toreduce the mortality and morbidity. As the pathogenic bacterium of TBM,Mycobacterium tuberculosis (MTB), hard to be stained by Gram stain becauseof the large amounts of mycolic acid consists in the wall of the bacterium,could be stained by acid-fast stain, as a type of acid fast bacterium (AFB).Once the AFB is found from the specimen of a patient’s cerebrospinal fluid(CSF), it is probably that the patient is affected by MTB. While the detectablerate of traditional acid-fast stain is low, the purpose of the modified acid-faststaining method we study on is to improve the detectable rate of AFB. Basicfuchsin, component of dyestuff in acid fast stain, has the characteristics of autofluorescence. When observed by fluorescence microscope, basic fuchsin,bounding to the nucleic acids of AFB, appears orange-red emission and easilyto be discovered. Modified acid-fast staining method is benefit to thediagnosis of TBM as an efficient, simple, frequent and inexpensive method.Methods: Collecting medical records of clinical suspicions of TBM,score them according to the latest TBM clinical diagnostic criteria, screeningout of51cases: the definite TBM1case, probable TBM15cases, possibleTBM30cases, impossible TBM5cases, include126CSF specimens. Thematched group includes36cases of non-tuberculous meningitis patients(non-TBM group) includes meninges carcinoma12cases, purulent meningitis5cases, cryptococcosis meningitis4cases, central nervous system leukemia4cases, Guillain-Barre syndrome5cases, cerebral cysticercosis2cases,multiple sclerosis2cases, viral meningitis1cases, brucellosis1cases. All of the patients should be taken the lumbar puncture at least one time, to obtainthe CSF specimens. The specimens, both experimental and matched group,should be stained by modified acid-fast stain, record the results and have ananalysis.Results:1The morphology of AFB found in modified-acid fast stain: Observedunder light microscope, AFB appears light red and rod-shape, some curved orhave a branch, some are more shorter, the length is2~5μm, and the diameter is0.3~0.5μm, most of them appears form a pile, as extracellular bacterium. Wealso find intracellular bacteria, located in neutrophils, monocytes andlymphocytes. While the cells in background and non-acid fast bacteria appearlight blue. Observed under fluorescence microscope, AFB appears orange-redemission, the morphology and size of AFB is the same as observed under lightmicroscope, while the background is extremely dark. In some fields, moreAFBs are found than light microscope.2The detection of AFB between experimental and matched group:44in51cases are detected AFB under both light microscope and fluorescencemicroscope in experimental group, the detectable rate is86.27%.5cases aredetected AFB under both light microscope and fluorescence microscope inmatched group, distribute in3cases of meninges carcinoma,1case of cerebralcysticercosis and1case of purulent meningitis, the detectable rate is13.89%.The differences of detectable rate between experimental group and matchedgroup are statistically significant (P<0.001). The sensitivity of modified-acidfast staining method is84.78%and the specificity is86.11%.3The comparison of light microscope and fluorescence microscope:44cases are positive in observation of both light microscope and fluorescencemicroscope, the detectable rate is86.27%.85in126parts of CSF specimensare positive under the observation of light microscope, the detectable rate is67.46%,1+:50specimens,2+:28specimens,3+:7specimens;90in126specimens of CSF specimens are positive under the observation offluorescence microscope, the detectable rate is71.43%,1+:49specimens,2+: 31parts,3+:10parts; the detectable rate between the two methods ofobservation has no statistically significant differences (P=0.359). The twomethods of observation have consistency (P<0.001).4The detectable rate of combine light microscope and fluorescencemicroscope:85in126CSF specimens are positive under light microscope, thedetectable rate is67.46%,90in126CSF specimens are positive under thefluorescence microscope, the detectable rate is71.43%, when light microscopecombined with fluorescence microscope,97in126CSF specimens arepositive, the detectable rate is76.98%, higher than light microscope orfluorescence microscope, the differences is statistically significant (P<0.001，P=0.016).5The detectable rate between single submitted cases and repeatedsubmitted case: Of the51cases in experiment group,26cases are singlesubmitted,25cases are repeated submitted. Under the light microscope,22cases are positive both in single submitted group and repeated submitted group,the detectable rate is84.62%and88.00%respectively. There is no significantdifference between the two group (P=0.523). Under the fluorescencemicroscope, the positivity of single and repeated submitted group is21casesand23cases, the detectable rate is80.77%and92.00%respectively. There isno significant difference between the two group (P=0.226).6The relationship between the accounts of cell in CSF and the positivityof modified acid-fast stain: According to the cell accounts, the CSF specimensdepart into two groups,8of14CSF specimens are positive in less than100group, the detectable rate is57.14%.72of101CSF specimens are positive inmore than100group, the detectable rate is71.29%. There is no significantdifference between the two group (P=0.218).7The relationship between the cerebrospinal fluid cytology (CSFC) andthe positivity of modified acid-fast stain: Of the experimental group,24in27CSF specimens are positive in neutrophils group, the detectable rate is88.89%;18in22CSF specimens are positive in mixed group, the detectable rate is81.82%;29in51CSF specimens are positive in lymphocytes group, the detectable rate is56.86%; the detectable rates of the first two groups arehigher than the third group, and the difference is statistically significant(P=0.035，P=0.004).8The detectable rate of each group in experimental group: Of the51cases,1case belongs to the definite TBM group, and outcome of the acid-faststain is positive;12of15cases are positive in probable TBM group;26of30cases are positive in possible TBM group;5cases are all positive inimpossible TBM group. In the126parts of CSF specimens,2of3CSFspecimens are positive in definite TBM group;36of51CSF specimens arepositive in probable TBM group;42of65CSF specimens are positive inpossible TBM group;5of7CSF specimens are positive in impossible TBMgroup. There is no significant difference of each group whether analyzed bycases accounts or CSF specimens (P=0.692, P=0.915).Conclusion:1Whether observed by light microscope or fluorescence microscope,modified acid-fast stain could obviously increase the detectable rate of AFB inCSF specimens compared with traditional acid-fast stain, its sensitivity is84.78%and the specificity is86.11%. The modified acid-fast stain, as amethod for screening TBM, is very helpful, should combined with otherexaminations to definite TBM.2There is no significant difference between light microscope andfluorescence microscope to detect AFB, while using modified acid-fast stain.The two methods of observation have good consistency. The detection of AFBis much more than light microscope or fluorescence microscope, whencombined the two methods.3There is no significant difference of the detectable rate between singlesubmitted cases and repeated submitted case.4There is no significant difference between the accounts of cell in CSFand the positivity of acid fast stain, while there has difference between the CSFC and the positivity of acid-fast stain.