Study About The Influence Of Pyrazinamidase Activity On The Clinical Efficacy Of Pulmonary Tuberculosis

BackgroundPyrazinamide(PZA)is a crucial first-line antituberculosis drug,which can kill nonreplicating persisters in acid environment or inside macrophages that are not killed by other drugs.It plays a unique role in shortening the TB treatment period and decreasing recurrence rate.The mode of action of PZA is unusual,it has strong sterilizing activity in vivo,but is active only at an acid medium in vitro.Meanwhile,PZA is a prodrug that is converted to the active form pyrazinoic acid(POA)by pyrazinamidase(PZase)in mycobacterium tuberculosis.The high-efficient activity of PZase is essential to PZA action.PZA will not converted to the active form pyrazinoic acid(POA)if PZase loss activity.PZA resistance is increasingly serious with its widely application.The current study found that mutations in the pncA gene encoding PZase are the major mechanism of PZA resistance.ObjectivesTo study the influence of pncA gene mutations and PZase activity on the clinical efficacy of pulmonary tuberculosis PartⅠ A cohort study about the influence of pyrazinamide resistance on the clinical efficacy of primary pulmonary tuberculosis.Subjects and methodsSubjects: The 75 patients with primary pulmonary tuberculosis and pyrazinamide resistance were selected as pyrazinamide resistance(PZA-R)group.Use 1:1 matching principle,recruited 75 patients with pyrazinamide sensitive and aged within 10 years of difference from the PZA-R patients as pyrazinamide sensitive(PZA-S)group。All patients of PZA-R group and PZA-S group had completed the treatment with a combination regimen include pyrazinamide and frozen their mycobacterium tuberculosis isolates in Guangzhou Chest Hospital from July 2012 to January 2016.Methods: Adopt the method of retrospective cohort study,We collected the general informations,radiological features,sputum results before and after treatment of two groups.Then compared the rate of sputum negative conversion,lesions absorption,cavity shrinking after 2 months and 6 months treatment between PZA-R group and PZA-S group.Results1.After two months treatment,the sputum negative conversion rate of PZA-R group and PZA-S group was 76.00%、85.33%,respectively(c2=2.095,P=0.148);the lesions absorption rate was 81.33%、88.00%,respectively(c2=1.284,P=0.257);the cavity shrinking rate was 43.14%、60.38%,respectively(c2=3.085,P=0.079).2.After six months treatment,the sputum negative conversion rate of PZA-R group and PZA-S group was 85.33%、93.33%,respectively(c2=2.519,P=0.113);the lesions absorption rate was 89.33%、94.67%,respectively(c2=1.714,P=0.190);the cavity shrinking rate was 54.90%、71.70%,respectively(c2=3.162,P=0.067).Part II The influence of pncA gene mutations on the clinical efficacy of pulmonary tuberculosis.Subjects and methodsSubjects: The 150 mycobacterium tuberculosis isolates of PZA-R group and PZA-S group that were frozen in Guangzhou Chest Hospital from Part I,each group had 75 isolates.Methods: Resuscitated and cultivated the above 150 isolates,their pncA genes were amplificated and detected by PCR-DNA sequencing technology.According to the pncA gene mutation or not,the patients were divided into two groups.The patients with PZA resistance and whose isolates carried pnc A gene mutation were divided into mutation group,and the matched patients with PZA sensitive and whose isolates without pncA gene mutation were divided into wild-type group.Then compared the rate of sputum negative conversion,lesions absorption,cavity shrinking after 2 months and 6 months treatment between mutation group and wild-type group.Results1.Among the 150 frozen isolates,we successfully resuscitated and cultivated 141 isolates,67 isolates were PZA resistant strains and 74 isolates were PZA sensitive strains.2.Among the 67 PZA resistant isolates,16 isolates had pncA mutations,but the 74 PZA sensitive isolates,only 1 isolates had pncA mutations.The mutation frequency of PZA resistant and PZA sensitive isolates was 23.88%、1.35%,respectively(c2 =16.832,P=0.000),which revealed pncA gene mutation associated with pyrazinamide resistance(r=0.327).3.Each has 16 cases of mutation group and wild-type group.After two months treatment,the sputum negative conversion rate of mutation group and wild-type group was 68.75%、87.50%,respectively(P=0.394);the lesions absorption rate was 75.00%、93.75%,respectively(P=0.333);the cavity shrinking rate was 41.67%、69.23%,respectively(P=0.238);the effective rates were similar between two groups.4.After six months treatment,the sputum negative conversion rate of mutation group and wild-type group was 81.25%、100.00%,respectively(P=0.226);the lesions absorption rate was 87.50%、100.00%,respectively(P=0.484);the cavity shrinking rate was 50.00%、84.62%,respectively(P=0.097);the effective rates were similar between two groups.Part III The influence of pyrazinamidase activity on the clinical efficacy of pulmonary tuberculosis.Subjects and methodsSubjects: The successfully resuscitated and cultivated 141 isolates in Part II,67 isolates were PZA resistant strains and 74 isolates were PZA sensitive strains.Methods: pyrazinamidase(PZase)activity were detected by the Wayne method for the above 141 isolates.Analysed the correlation among PZase activity and PZA susceptibility,pncA mutation.Then,the patients with PZA resistance and PZase negative were divided into negative group,and the matched patients with PZA sensitive and PZase positive were divided into positive group.Then compared the rate of sputum negative conversion,lesions absorption,cavity shrinking after 2 months and 6 months treatment between negative group and positive group.Results1.Among the 67 PZA resistant isolates,28 isolates were negative for PZase,39 isolates were positive.Among the 74 PZA sensitive isolates,4 isolates were negative for PZase,70 isolates were positive.The PZase negative rate of PZA resistant and PZA sensitive isolates was 41.79%、5.41%,respectively(c2=26.534,P=0.000),which revealed pyrazinamide resistance associated with the loss of PZase activity(r=0.398).2.Among the 32 PZase negative isolates,17 isolates had pncA gene mutations;No mutations existed in the 109 PZase positive isolates.The mutation frequency of PZase negative and PZase positive isolates was 53.13%、0.00%,respectively(c2=60.930,P=0.000),which revealed pncA gene mutation associated with the loss of PZase activity(r=0.564).3.Each has 27 cases of negative group and positive group.After two months treatment,the sputum negative conversion rate of negative group and positive group was 77.78%、85.19%,respectively(c2=0.491,P=0.484);the lesions absorption rate was 77.78%、88.89%,respectively(c2=0.533,P=0.465);the cavity shrinking rate was 42.86%、68.18%,respectively(c2=2.794,P=0.095);the effective rates were similar between two groups.4.After six months treatment,the sputum negative conversion rate of negative group and positive group was 85.19%、100.00%,respectively(c2=2.430,P=0.119);the lesions absorption rate was 88.89%、100.00%,respectively(c2=1.412,P=0.235);the cavity shrinking rate was 57.14%、81.82%,respectively(c2=3.101,P=0.078);the effective rates were similar between two groups.Conclusions1.Primary pulmonary tuberculosis patients with pyrazinamide resistance were treated with a combination regimen include pyrazinamide,whose clinical efficacy were similar to pyrazinamide sensitive patients.2.pncA gene mutation associated with pyrazinamide resistance.3.Primary pulmonary tuberculosis patients with pncA gene mutation were treated with a combination regimen include pyrazinamide,whose clinical efficacy were similar to those without pncA gene mutation.4.pyrazinamide resistance associated with the loss of PZase activity.5.pncA gene mutation associated with the loss of PZase activity.6.Primary pulmonary tuberculosis patients with PZase negative were treated with a combination regimen include pyrazinamide,whose clinical efficacy were similar to PZase positive patients.