Research On The Cerebrospinal Fluid Cytology And The Susceptibility Gene (TLR2T597C) Of Tuberculous Meningitis

ObjectiveTuberculous meningitis (TBM) is the most severe form of tuberculosis (TB) killing20-25%of those affected and leaving neurological sequelae in many of those who survive. High mortality and morbidity partly due to the delayed diagnosis and treatment, therefore improve the early diagnosis of TBM become one of the urgent problems in diagnosis and treatment of TBM. It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), Among those putatively infected only around10%will ever develop clinical disease, and TBM accounted for around1%of all cases. Why some patients develop TBM, but most do not, the reason is not clear. There may be host genetic, environmental or bacterial factors that make patients more susceptible to this form of the disease.The diagnosis of TBM is difficult because of the atypical clinical manifestations and lacking of rapid and sensitive diagnostic method, missed diagnosis and delayed treatment result in significant mortality and morbidity. The incidence of TBM is significantly higher in some countries and regions, where the standard of living is low, the living environment is poor, while the acid-fast staining as one of the gold standard for diagnosis of TBM is an ideal diagnostic method because of time-consuming shorter and inexpensive equipment and reagents required. However, different studies reported positive rates greatly different, and affected by the drug treatment, after5-15days drug treatment, the positive rate can be reduced from52%to2%. So, we need to analyze some indicators which easy to obtain in clinical to predict the acid-fast positive ratio, and improve the diagnosis of TBM.We found that the CSF cytology performance of TBM patients who diagnosed by etiology is different in different patients, the same patients in different periods also have different CSF cytology performance, therefore, we conclude this may be related to the susceptibility of patients. Health Center of McGill University in Canada found that there are around2billion people are infected with Mtb, but the final incidence of less than10%, while the TBM accounted for very small part of it, these results strongly suggested that some individuals are susceptible to TBM. Many studies have reported the association of T597C polymorphisms of TLR2gene with susceptibility to TB, but none of them research the susceptibility to TBM in han population of southern China.Out study was to analyze the cerebrospinal fluid cytology, conventional and biochemical test results finding out the meaningful indicator for predicting the proportion of acid-fast stain positive, and to seek a conventional solution to improve the level of TBM early diagnosis, our study also through the case control studies, reveals relationship between T597C single nucleotide polymorphisms(SNP) of TLR2with TBM in han population of southern China, provide important basic data for the disease correlation and genetic evolution research.Methods334CSF specimens come form suspected TBM patients who treatment in Nanfang Hospital from September2009to July2011was collected, according to the TBM diagnostic criteria developed in2010by the International Organization, choose83CSF specimens witch come from patients who meet the diagnostic criteria, addition to28CSF specimens witch come from the patients who suffering from nausea neoplasms, HIV infection or autoimmune disease, removal of18CSF specimens witch miss the conventional test items, removal of7CSF specimens witch PCR is positive, remaining30CSF specimens included in the study, divided into acid-fast positive group and the acid-fast negative group. there are TBM, tuberculosis and normal control group in the study of susceptibility gene.Blood and Cerebrospinal fluid (CSF) samples were collected from9HIV-negative adult patients with TBM between September2009to May2011from Nanfang Hospital in Guangzhou, China. All patients with TBM must meet the diagnostic criteria developed by the international tuberculous meningitis workshop in2010. they must have one or more Symptoms and signs of meningitis as following: headache, irritability, vomiting, fever, stiff-neck, convulsions, focal neurological defects, altered consciousness, or lethargy, and combine one or more of the following: acid-fast bacilli seen in the CSF; Mtb cultured from the CSF; or a CSF positive commercial nucleic acid amplification test. Blood samples from20patients with pulmonary TB were collected in December2011from Guangzhou Chest Hospital. All patients had’uncomplicated’pulmonary TB, defined as no clinical or radiographic evidence of miliary or extrapulmonary TB and acid fast bacilli in sputum and/or the culture of M. tuberculosis from sputum. The control group came from unrelated healthy individuals who were ethnic Chinese. All Objects participant in this study were han population come from southern China, and exclude tumor, HIV infection, autoimmune diseases. Written informed consent was obtained from each objects participant in this study.Collection of CSF in study of CSF cytology, part sent to the clinical laboratory,the rest sent to the department of neurology for MGG staining and Ziehl-Neelsen acid-fast staining. Peripheral blood (anticoagulant tube) and CSF specimen were collected from all the participants, quick-frozen in liquid nitrogen, and then stored in-80℃refrigerator. Extracted genomic DNA from the blood and CSF using the QIAamp DNA Blood, According to the information about TLR2(rs3804099) provided by the GenBank database, design primers using Oligo6.0or premier5.0software for the PCR amplification of TLR2gene translation initiation site in the coding region, sense primer:5’-TTTTCTTCCCTGGGCAGT C-3’; anti-sense:5’-TCGCAGTTCCAAACATTCC-3’; the length of the PCR a mplification product was400bp. The products were sent to Guangzhou Genomi c Center (BGI) for gene sequencing. Compared sequencing results with the info rmation of TLR2in the BLAST database, obtained TLR2gene single nucleotide polymorphisms (SNPs).SPSS13.0statistical software was used for the data processing. CSF cytology studies using independent samples t-test screening of acid-fast stain positive factors that may affect, Logistic regression analyze acid-fast stain positive impact factors, using the Roc curve to establish the predictive value of the critical value;in study of susceptibility genes, t-tests were used for the Measurement data, expressed by the form of (X±s). Chi-squared tests were used for the comparison of allele and genotype distribution in the study groups. The Hardy-Weinberg equilibrium test was assessed in control group. A p-value of <0.05was accepted as significant difference.ResultsIndependent samples t test found that only the lymphocytes percentage, monocytes percentage, neutrophils percentage, eosinophils percentage, plasma cells percentage and CSF glucose and blood glucose ratio between the two groups(Acid-fast stain positive group and negative group) have significantly different. Logistic regression found that only neutrophils percentage, plasma cells percentage and CSF glucose and blood glucose ratio into the regression equation. The ROC curve results show that:when the percentage of neutrophils≥37.5%, the sensitivity for acid-fast stain positive is80%, specificity65%; when the percentage of plasma cells≥2.5%, sensitivity80%, specificity85%; when CSF glucose and blood glucose ratio≥0.285, sensitivity70%, specificity80%; regression equation is logit(P)=β0+β1x1+β2x2+...++βpxp when P values≥.3977654, sensitivity80%, specigicity90%. The study of susceptibility gene included9patients of TBM,20patients of pulmonary tuberculosis, and20healthy controls. Their mean±SD age was32.7±12.1years in TBM group,38.3±14.8years in pulmonary tuberculosis group, and25.0±2.4in control group. In the TBM group,2(22.2%) were male and9(77.8%) female, In pulmonary tuberculosis group and control group, the male to female ratio was1:1. TBM and pulmonary tuberculosis group has no significant differences in age and sex composition. Control group comply with the Hardy-Weinberg equilibrium test(χ2=0.726, P=0.394>0.05), come from a genetic equilibrium population.Since the small sample size, the frequency of597CC genotype is low, we called597TC,597CC genotype as variant genotypes. The frequency of variant genotypes in the control group,the pulmonary tuberculosis group and TBM group were55%(11/20),65%(13/20),89%(8/9) respectively, have no significant difference among the three groups(x2=3.560, P=0.169), also between the tuberculosis group witch contains the pulmonary tuberculosis and TBM group and control group (χ2=1.584, P=0.208,OR=2.15,95%CI:0.65-7.13). The frequency of597C allele in the control group, pulmonary tuberculosis group and TBM group were30%(12/40),38%(15/40),61%(11/18) respectively, also have no significant difference among the three groups (χ2=5.108, P=0.078), also between the tuberculosis group and control group(χ2=2.192, P=0.139, OR=1.90,95%CI:1.81-4.44). The frequency of the variant genotypes (T/C and C/C) and597C allele in TBM group were much higher than in the control group, but due to the lower test power (power<0.5), have not yet obtain a statistically significant difference.ConclusionIn CSF cytology study, we found that the increased percentage of neutrophils and plasma cells, the decreased CSF glucose and blood glucose ratio are related with the positive acid-fast staining, through the CSF cytology, conventional biochemical test results, predict the acid-fast stain-positive rate, improve the predictability of cliniciansin for the TBM diagnosis. However, due to the limited number of specimens, some indicators witch may be related to acid-fast positive not yet reached a statistically difference. Found in the CSF cytology dynamic observation of patients confirmed TBM that the same patients in different periods have different performance,this suggest that a variety of inflammatory genes involved in different disease process in TBM. We assume that there is a causal relationship in the changes of these genes, the performance of CSF cytology and the susceptibility to TBM, and part of the gene belongs to the TBM susceptibility genes. Therefore, we searched the previous research literature, and conducted a preliminary screening of the CSF gene chips, selected TLR2gene as the research targets. We reported the association of T597C polymorphisms of TLR2gene with susceptibility to tuberculosis especially to TBM in han population of southern China.TLR2is capable of recognizing PAMPs expressed by Mtb, such as a lipoarabinomannan,19-kDa lipoprotein, and soluble tuberculosis factor. When TLR2combined with the lipoproteins of Mtb, it can induce the apoptosis of human macrophages which considered to be an important mechanism for killing the Mtb within the macrophages. The distribution of T597C SNP of TLR2was significantly different in different ethnic and regions. In our study, the frequency of the597T allele in normal population was70%(28/40), but3%(3/110) in health Caucasian population,75%(564/754),50%(24/48) in health population of Vietnam and Japan respectively. This shows that597T allele is a minor gene for Caucasian population, but a common gene for Asian population, these differences may affect the function of the gene.A study conducted by Thuong and his colleagues in Vietnam, including183cases of pulmonary tuberculosis,175cases of TBM, and389umbilical cord blood as control, they found that TLR2SNP T597C is associated with susceptibility to tuberculosis (all tuberculosis vs control,OR=2.22,95%CI:1.23-3.99), then they compared the allelic and genotypic frequencies of SNP T597C in these two groups to the control group frequencies. they found no association between SNP T597C and pulmonary TB, but have significant association with TBM (TBM vs control,OR=3.26,95%CI:1.72-6.18), especially significant in patients complicated with evidence of military tuberculosis (TBM+military tuberculosis vs control, OR=5.28,95%CI:2.20-12.65), and also associated with severity of TBM at the start of treatment. In addition, they also found that Co-inheritance of TLR2SNP T597C and TIRAP SNP C558T increases susceptibility to TBM. Maxine Caws and his colleagues also reported the TLR2SNP T597C is associated with TBM, rather than pulmonary TB, researched in Vietnam, including237cases of pulmonary tuberculosis,187cases of TBM, and389umbilical cord blood as control, but this association only with TBM infected with Beijing lineage. In order to study the association of T597C SNP with tuberculosis, especially to TBM in the southern Chinese Han population, we adopt case-control study, and found that the frequency of variant genotype (TC, CC) and597C allele were significantly higher in TBM group than in control group, T597C SNP and tuberculosis especially TBM, may have association, this result consistent with the findings discovered by Thuong and Maxine Caws in Vietnam. However due to limited by the number of test cases, the test power is low (power<0.5), have not yet obtain a statistically significant difference.The initial point of tuberculosis infection is entry of the bacilli into the lungs via inhalation of infectious droplets, whereupon the bacteria colonize macrophages within the alveoli, and begin to copy. bacteria may disseminate to local lymph nodes and bloodstream, before the start of the acquired immune, bacteria can spread through the systemic circulatory system to other organs, leading to extrapulmonary tuberculosis, such as TBM. This process affected by the innate immune response, and by the interaction of TLR2and Mtb. Studies done by Thuong and Maxine Caws in Vietnam confirmed this viewpoint, in our study variation genotype and597C allele were higher in TBM group than in control group also supported this view from the other side.In summary, our study found increased neutrophil and plasma cells proportion, decreased CSF glucose and blood glucose ratio have important prompt role for acid-fast stain positive,simultaneously. through the dynamic observation of CSFcytol ogyof TBM patients speculated the exist of genetic susceptibility. But our study still can not prove the association of TLR2SNP T597C with susceptibility to tuberculosis and TBM in han population of southern China, however variation genotype and597C allele were significantly higher in TBM group than in control group,, if we increase the number of cases, improve the test power, maybe we can find the association of TLR2SNP T597C with susceptibility to tuberculosis or TBM. Since Nanfang hospital is a first class hospital in Guangzhou, most patients suspected of suffering TBM have been given drug therapy, at this time, positive rate is very low of detection, Mtb from CSF, so a small number of patients enrolled, test power is low, reflected limited information, failed to reach a statistically significant difference, so a large sample size research need to be done. In addition, the host susceotible to Mtb infection is determined by multiple genes, the role of each gene is limited, the exact mechanism of susceptibility or protection is controversial. It is noteworthy that whether the one who carry the heterozygous genotype but not suffer from TBM now will developed into TBM, it requires a long-term follow-up study.