The Culture And Cytological Analysis Of Mycobacterium Tuberculosis From Tuberculous Meningitis Cerebrospinal Fluid

Background:Tuberculous meningitis (TBM) is the most severe form of tuberculosis andcauses substantial morbidity and mortality in children and adults. Delayed ormissed diagnoses contribute to Mycobacterium Tuberculosis (MTB)transmission and mortality due to TBM. However, the early and accuratediagnosis of TBM is still a challenge for clinicians not only because of itsdiverse clinical manifestations but also because of the current lack of rapid andsensitive detection methods. A number of options are currently available for therapid diagnosis of TBM. Mycobacterial culture is the diagnostic gold standardfor detection of tubercle bacilli but it is time-consuming and requires specializedsafety procedures in laboratories. Serological method is convenient but lackssensitivity and specificity. Polymerase chain reaction technique is rapid butcostly to be routinely used in developing countries where most TB cases present.The conventional smear microscopy with Ziehl-Neelsen staining is a maindiagnostic method for detection of acid-fast bacilli, especially in the low-income countries, due to its rapidity, low cost, and high positive predictive value for TB.However, the major disadvantage of Ziehl-Neelsen staining method is lowdetection rate, ranging from0–20%from the CSF specimens. In experiencedhands, using large CSF volumes (>6mls) and meticulous examination of slides(at least30minutes) the sensitivity of smear can exceed60%. One possiblereason beneath low sensitivity could be due to Mtb cytozoic characteristic, thatis, once entering the cells, Mtb is hardly stained by acid-fast dyes. Anotherreason is due to the detection limit of Ziehl-Neelsen staining method, unable todetect the bacilli whose numbers are fewer than104per slide or per ml ofspecimen.The BD BACTEC MGIT960System is the new generation in aproven line of mycobacteria testing instruments from BD. The BACTEC MGIT960System builds on the legacy of simplicity, efficiency, performance andsafety of the BACTEC460TB and9000MB instruments. MGIT stands forMycobacteria Growth Indicator Tube, and960indicates the total number ofculture tubes it can hold at any given time. The BACTEC MGIT960System isan in vitro diagnostic instrument for rapid detection of Mycobacteria in clinicalspecimens other than blood. The system is designed to meet the needs ofmedium and high volume labs, capable of processing about8,000cultures peryear. This system is simple, efficient, safe to use and occupies small laboratoryspace.Methods:1. MGIT960mycobacterial cultureThe BD BACTEC MGIT960System is the new generation in aproven line of mycobacteria testing instruments from BD. The CSF of suspectedTBM was culture according MGITTMProcedure Manual.2. Cerebrospinal fluid cytologyThe CSF specimens were centrifugated by FMU cytospin and stained byMGG (May-Grunwald Giemsa) method for10min. 3. Modified Ziehl-Neelsen stainingThe modified Ziehl-Neelsen staining was performed as we described before.In brief, Cytospin was used to collect the formed elements of CSF specimens.0.5ml CSF specimen was loaded into the chamber of a cytospin, in whichpoly-l-lysine-coated slides were inserted, and centrifugated at1000g for5-10min. After aspirating media, the cells were fixed with4%paraformaldehyde for15min at room temperature. Before the Ziehl-Neelsen staining, the cytospincells were permeabilized with0.3%TritonX-100for30min. During staining,the fast acid dye containing0.3%TritonX-100was used.Results:The MGIT960method significantly improved the Mycobacteriumtuberculosis culture positivity in the CSF of TBM patients, and there typicalcerebrospinal fluid cytology was lymphocyte predominant (almost60%) orneutrophil predominant (almost1/3)。The CSF WBC number was significantlyhigher in the first week after the onset and almost equal in the other time point.There were plasm cells present in the CSF of culture proved TBM patiants. The8of37culture proved TBM patians were both positive by conventional andmodified ZN staining while all these37culture proved TBM patians were onlypositive by modified ZN staining. Except for the extracellular isolated Mtb,there were many intracellular Mtb present within the monocytes、neutrophil andlymphocytes by modified ZN staining. As more and more culture proved TBMwere confirmed by MGIT960method and Mtb were deteced by modified ZNstaining, the precise mechanisms and typical clinical manifestation of TBMpatients will be uncovered.ConclusionOur study found that improved fight acid dyes greatly improved thedetection rate of cerebrospinal fluid acid fast bacilli. Discovered tuberculosisbacterium of cerebrospinal fluid cell firstly. Discovering the lymphocyte has swallowed tuberculosis bacterium function firstly. Improved fight acid dyesmethod is simple, rapid and efficient. Basic-level hospital in an acid can beimproved dyeing method which can substitute the traditional fight acid dyes.