| Objective:To establish a new approach for the quantitative detection of VAD1 mRNA of cryptococcus neoformans (CN) by real-time fluorescent quantitative PCR (RT-FQ-PCR), and appraise its value in the evaluation of the treatment efficacy of patients who suffered from cryptococcus neoformans meningitis (CNM). In a meanwhile, the Th1/Th2 cytokines of patients were assayed using ELISA, to investigate the potential contribution of VAD1 expression in the alteration of cytokine profiles.Methods:1. The primers and TaqMan probe were designed according to the published sequence of VAD1 mRNA (GenBank AY-661864) for the establishment of RT-FQ-PCR. The recombinant vector pGEMT-Easy-VAD1 was used as the standard template and then a standard curve was established. The PCR reaction system was optimized and evaluated.2. Cerebrospinal fluid from 25 CNM patients and 30 controls were diagnosised by RT-FQ-PCR, India ink staining, fungus culture and cryptococcal capsular antigen quantitation. The sensitivity and specificity of the four methods were compared byχ2 test.3. The VAD1 mRNA concentration of CSF from both pretherapy and post-therapy of 25 CNM patients were also detected, combined with the related clinical manifestation, we apparise its clinical significance for the evaluation of the treatment efficacy.4. We quantitated the IFN-γ,IL-12,IL-10 cytokines concentration in the CSF of 25 CNM patients, and analyzed the relationship between the concentration of VAD1 mRNA and these cytokines. Results:1. The RT-FQ-PCR method for the quantitation of the VAD1 mRNA have been established successfully. Its detection low limit is 101 copies/ul, the correlation coefficient of the standard curve is -0.9979. The CV values for high, medium and low concentrations were 0.65%, 0.89% and 1.23% respectively, and the sensitivity was 96% when its specificity was 100%.2. The sensitivity of RT-FQ-PCR is 96% and its specificity is 100% in the diagnosis of CN. The sensitivity of RT-FQ-PCR is higher than that of ink staining method and culture method (p<0.05, p<0.05 respectively). There is no difference in the sensitivity between RT-FQ-PCR and antigen detection method in the detection of CN (p>0.05).3. VAD1 mRNA concentration in acute phase of CNM were higher than that of in recovery phase patient (3.042±0.906 Vs 2.187±0.665, P<0.01). The levels of VAD1 mRNA in CNM patients were positively correlated with the amount of CN (r=0.870,P<0.01) and intracranial pressure (r=0.770,P<0.01), negatively correlated with Glucose concentraqtion(r= -0.429, P<0.05). The level of VAD1 mRNA was decreased significantly in the group of patients received AmB, 5-FC and FZC compared to patients used 5-FC plus AmB or 5-FC plus FCZ. VAD1 mRNA expression was reduced the most in patients treated with AmB+5-FC+FCZ. VAD1 mRNA decreased in three treatment groups did not show significant differences (p>0.05)4. The levels of VAD1 mRNA were closely related to IFN-γ, IL-12 and IL-10. 25 CNM patients had lower rations of IFN-γ/ IL-10 or IL-12/ IL-10 compared to control group, and the rations in acute phase are significant higher than stable phase. The levels of VAD1 mRNA were negatively correlated with IFN-γ, positively correlated with IL-10, and did not show correlation with IL-12.Conclusions The established RT-FQ-PCR method for the detection of VAD1 mRNA exhibits a good sensitivity, specificity and reproducibility, it could be used to measure quantitatively the severity of the infection, and could be used in the assessment of therapy response and prediction of prognosis. The expression level of VAD1 mRNA is related to the treatment efficacy of CNM. CNM was considered a dominant Th2 response,and convalescent period Th1/ Th2 shift to Th2, suggests that Th dysregulation may contribute to the pathogenesis of cryptococcosis.
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