To Explore The Valuation And Influenced Factors Of The Binary PCR In Diagnosis Of Tuberculous Meningitis

Objective: Tuberculous meningitis (TBM) is a central nervous systeminfection disease caused by Mycobacterium tuberculosis(MTB), one of themost serious type in tuberculosis, its fatality rate and disability rate is veryhigh. The incidence of TBM was increasing in recent years due to variousfactors, such as drug-abuse, the desending immunity cause of many reasons.Fast diagnosis and promptly treatment is the key to affect prognosis. But thediagnosis of the TBM, especially early diagnosis is difficult. According to thestatistics,the incidence of TBM was increasing in recently15years of thesecond hospital of Hebei medical university. Finding mycobacteriumtuberculosis from Cerebrospinal fluid（CSF）by culture, Z-N stain andpolymerase chain reaction(PCR) can be used as "gold standards" fordiagnosing TBM, but the positive of smeared an acid fast stain or culturedtuberculosis mycobacterium is very low, therefor their value for earlierdiagnosis in clinical is limited. At present there is still no a reliable andpractical diagnostic method to detect MTB can satisfy the need of clinicaldiagnosis. PCR is mature, many researchers did more works about MTB-PCRfrom CSF, concluding that PCR had high valuation in diagnosing TBM. Toincrease the detected rate of MTB from CSF, we use binary PCR to detectMTB from CSF and analyse the sensitivity and specificity. Then by analysingthe relationship between PCR and the course of treatment、CSF cytology、protein、glucose or chloride, we can know the practical value.Methods：The case group were50cases （79shares of CSF）clinicaldiagnosis of tuberculous meningitis patients, and the control group were30cases（30shares of CSF）not tuberculous meningitis patients. All of thepatients were examined CSF routine, biochemical, cytology(MGG dye) andmodified Ziehl-Neelsen(Z-N) stain, meantime detected special target DNA of MTB complex by using binary PCR. Collecting1mL CSF and then reservingin-80°refrigerator. We melted the CSF in room temperature and extract MTBDNA from CSF using the kit for extracting DNA of yeast. Weelectrophoresised by2%sepharose gel. The positive result is that we found theband of123bp or239bp, other result is negative. By comparing PCR positiverate between the two groups and the relationship between PCR and the courseof treatment、CSF cytology、protein、glucose、chloride or the result of modifiedN-Z stain, we analyse the positive rate and specificity of the binary PCR, andexplore the influnced factors of PCR to give the theoretical basis for diagnosisTBM in clinical work.Results:1Clinical data: Tuberculous meningitis patients might manifest as fever,headache, and we also find meningeal stimulation sign, brain focal signs,consciousness obstacleetc etal, but it lack of specificity, not easy to identifywith other diseases of the nervous system.2Cerebrospinal fluid routine and biochemical examination results: Thecase group, the average number of white blood cells count were70（194）×10~6/L; the average glucose were2.22（1.25）mmol/L；protein’s average were1.04（1.07）g/L；chloride’s average were117.8（12.5）mmol/L; the averagepressure of lumbar puncture were240（151.75）mmH2O. In the control group,white blood cells132.37±293.46×10~6/L；glucose2.91±0.95mmol/L；protein0.76±0.79g/L；chloride119.25±6.76mmol/L；the pressure of lumbar puncture225.56±122.38mmH2O.3The performance of cerebrospinal fluid cytology: From the entirecourse of desease, there are35cases(70%) in50cases presenting a typicalcytology of TBM. Neutrophil reaction in exudative phase, transiting quickly tomixed cytology, or neutrophil dominance of mixed cytology in exudativephase and lymphocyte dominance of mixed cytology in proliferative phase,during treatment for4-8weeks, lymphoidocyte reaction rehalf of recoveryphase is coming. At last, the cytology will be normal. The other15cases werealways presenting lymphoidocyte reaction and recover normal after treatment. In control group of30cases, there are18cases presenting normal cytology,1case presenting blooding cytology and11cases presenting stimulatedmonocyte-increased reaction meantime found tumor cells in cytology. We alldid not find cryptococcus in cytology.4The detection result of MTB PCR from CSF: Positive results by thebinary PCR were found in39adults of50patients with tuberculous meningitis,and5positive in30adults from the control group. The sensitivity andspecificity of the binary PCR for MTB respectively are78.00%and83.33%.Two groups were compared, P <0.05, the difference was statisticallysignificant. Positive results by the binary PCR were found in51shares of79shares of the paitents with tuberculous meningitis, and5positive in30sharesof the paitents from the control group. The sensitivity and specificity of thebinary PCR for MTB respectively are64.56%and83.33%. Two groups werecompared, P <0.05, the difference was statistically significant.5the comparison between the binary PCR of MTB and modified N-Zstain in the case group: There were51of79shares CSF from the case groupstained by modified N-Z stain. We found that25shares CSF in41shares Stainpositive were positive by PCR testing. And meantime, We found that1sharesCSF in10shares Stain negative were positive by PCR testing. Two methodswere compared, P <0.05, the difference was statistically significant.6the relationship between the binary PCR of MTB and the course oftreatment in the case group: In the case group, there are62shares CSF frompaitents who were not treated or were treated in one months. The paitents whobegan being treated longer than one months offer17shares CSF,11sharesCSF among17shares were got from the paitents whose clinical manifestionand all examinations were changing better and the other6shares were gotfrom the paitents whose clinical manifestion and all examinations werechanging worse. Positive rates of the there groups were compared. Newtreated group were compared with the after-treated group, P＞0.05, thedifference was not statistically significant.7the relationship between the binary PCR of MTB and CSF cytological dynamic change: In the case group,we compare positive rates of the binaryPCR between15adults whose CSF cytology was not found neutrophils and35adults whose CSF cytology was found neutrophils, P＞0.05, thedifference was not statistically significant; in the paitents whose cytology werenot found neutrophils, compared positive rates of the binary PCR between26adults whose CSF neutrophil proportion was lower than70%and9adultswhose CSF neutrophil proportion was higher than70%, P＞0.05, thedifference was not statistically significant.8the relationship between the binary PCR of MTB and CSF protein: Inthe case group,we compare positive rates of the binary PCR between9adultswhose CSF protein was lower than and equal to0.40g/L and70adults whoseCSF protein was higher than0.40g/L, P＞0.05, the difference was notstatistically significant; in the paitents whose CSF protein was high, comparedpositive rates of the binary PCR between27adults whose CSF protein wasbelong to (0.40~1.0)g/Land43adults whose CSF protein was higher than1.0g/L, P＞0.05, the difference was not statistically significant.9the relationship between the binary PCR of MTB and CSF glucose: Inthe case group,we compare positive rates of the binary PCR between34adultswhose CSF glucose was higher than and equal to2.40mmol/L and45adultswhose CSF glucose was lower than2.40mmol/L, P <0.05, the difference wasstatistically significant; in the paitents whose CSF glucose was low, comparedpositive rates of the binary PCR between40adults whose CSF glucose wasbelong to (1.00~2.39)mmol/Land5adults whose CSF glucose was lower than1.00mmol/L, P＞0.05, the difference was not statistically significant.10the relationship between the binary PCR of MTB and CSF chloride: Inthe case group,we compare positive rates of the binary PCR between30adultswhose CSF chloride was higher than and equal to120.00mmol/L and49adults whose CSF chloride was lower than120.00mmol/L, P <0.05, thedifference was statistically significant; in the paitents whose CSF chloride waslow, compared positive rates of the binary PCR between42adults whose CSFglucose was belong to (102.00~120.00)mmol/Land7adults whose CSF glucose was lower than102.00mmol/L, P＞0.05, the difference was notstatistically significant.Conclusion: The binary PCR of MTB is mature and provide a sensitivemethod for the early diagnosis of TBM. Its sensitivity is78%. If we testCSF-MTB by binary PCR and then treat disease early, its fatality rate anddisability rate will significantly descend. Both the binary PCR and modifiedZ-N stain have high sensitivity, but positive rate of modified Z-N stain ishigher than the binary PCR. MTB-PCR of CSF is closely related to thedescending of CSF glucose and chloride, not related to CSF neutrophilproportion and the level of CSF protein. We should be active to test MTB afteranti-MTB for1month to exclude the drug resistant,then we could modify thetreatment promptly.