Establishment And Validation Of Pyrazinamide Susceptibility Testing Methods Of Mycobacterium Tuberculosis

Background: Pyrazinamide(PZA)is the backbone of the short-course chemotherapy of tuberculosis due to its unique ability of killing the semi-dormant tubercle bacilli that reside in acidic inflammatory environments.Due to its effectiveness and low price,PZA is widely used in the intensive phase of anti-TB treatment and recently has become pivotal in MDR-TB(Multidrug-resistant tuberculosis,MDR-TB,defined as resistance to at least isoniazid(H)and rifampicin(R))treatment.In recent years,various studies have reported PZA resistance among MDR and non-MDR TB patients,which highlights the importance of PZA susceptibility testing before it being administered to patients.However,detection of PZA susceptibility is not very common as compared with other anti-TB drugs.PZA is active only in acidic environment(optimal p H=5.5)thus it affects the growth of mycobacteria and then alleviates the reliability of the test.In this study,we establishedand evaluated several pyrazinamide susceptibility testing methods,and try to pursure a simple,reliable and feasible method for clinical use.Part 1: Detection of pyrazinamide resistance of Mycobacterium tuberculosis using nicotinamide as a surrogateObjective:Despite the importance of pyrazinamide(PZA)in tuberculosis treatment,PZA susceptibility testing is not routinely performed because of acid p H requirement.The susceptibility testing for PZA had been established using nicotinamide(NIC)as a surrogate in neutral p H,however,very limited studies had been done to evaluate or validate the method.The purpose of this assay was to evaluate the Microplate Alamar Blue assay(MABA)to detect resistance to PZA using NIC at neutral p H,and identify the appropriate cutoff point for the assay.Methods: The NIC minimal inhibition concentrations(MICs)for 125 Mycobacterium tuberculosis(MTB)clinical isolates were tested by MABA at nine different concentrations(from 8 to 2000μg/ml).The PZA susceptibility testing by BACTEC MGIT960 system was used as a reference method.pnc A gene and its promoter region were sequenced for all the recruited strains.Result:64 out of the 125 clinical isolates were identified as resistant by MGIT960.Using MIC >500 μg/ml,as the cutoff concentration to define resistance,presented the best fit of the MABA assay with the MGIT960 outcomes.MABA demonstrated sensitivity of 100%,specificity of 95.2% and an accuracy of 97.6% when compared with MGIT960 method.Nine PZA susceptible strains defined by MGIT960 also had pnc A mutations,however,three of them were defined as PZA resistant by NIC MABA with MIC ≥2000 μg/ml.When using pnc A gene sequencing as the reference method,MABA demonstrated sensitivity of 89.5%(51/57),specificity of 80.9%(55/68)and an accuracy of 84.8%(106/125).Conclusion : Nicotinamide substitution method for PZA susceptibility test is reliable,cheap,rapid,and easy to perform,which made it very promising to be used in clinical laboratoriesPart 2: Rapid detection of PZA resistance of mycobacterium tuberculosis by probe enzyme digestion using SurveyorObjective: Establishment and validation of a probe enzyme digestion method using Surveyor for rapid detection of PZA resistance detection pf MTB.Methods: 91 MDR strains were recruited.The probe enzyme digestion was conducted by adjusting the ratio of forward primer and the reverse primer in the PCR reaction,using the tesed strain and the reference straind H37 Rv both as templates.After PCR amplification,a hybridization reaction was performed,and then,incubated with Surveyor.The products were then undertook electrophoresis.If the product was cut by Surveyor and more than one band were observed on the gel,the strain would be considered as harboring mutation in pnc A or its’ promoter region.BACTEC MGIT960 method and gene sequencing method were used as reference methods.Results: According to the the BACTEC MGIT960 method,the sensitivity,specificity and accuracy of the probe enzyme digestion method was 100%(29/29),87.1%(54/62)and 91.2%(83/91),respectively.According to the the pnc A gene sequencing method,the sensitivity,specificity and accuracy of the probe enzyme digestion method was 100%(29/29),100%(54/54)and 100%(91/91),respectively.Conclusion: The probe enzyme digestion method demonstrated very hig concordance with pnc A gene sequencing method.As a fast,easy,cheap method,the probe enzyme digestion method can be used as an alternative method of gene sequencing for pnc A mutation dectection.Part 3: The reliability evaluation of Versa TREKMyco PZA kit for pyrazinamide resistance detection of mycobacterium tuberculosisObjective: To evaluate the reliability of Versa TREKMyco PZA kit for PZA resistance detection of mycobacterium tuberculosis.Methods: A total of 192 clinical isolates of mycobacterium tuberculosis were analyzed.The tests were performed according to the manufactures’ recommendations using BACTEC MGIT960 system and Versa TREKMyco PZA kit.pnc A gene and its promoter region were sequenced for all enrolled strains.Results: According to the the BACTEC MGIT960 method,the sensitivity,specificity and accuracy of the Versa TREK Myco PZA kit method was 91.5%(119/130),93.5%(58/62)and 92.2%(177/192),respectively.According to the the pnc A gene sequencing method,the sensitivity,specificity and accuracy of the Versa TREK Myco PZA kit method was 88.4%(61/69),82.9%(102/123)and 84.9%(163/192),respectively.Conclusion: Versa TREK Myco PZA kit had similar capability for PZA resistance dectction as that of BACTEC MGIT960 system.When compared with pnc A gene sequencing,the Versa TREKMyco PZA kit method has had a good sensitivity.Part 4:The reliability evaluation of Wayne method for detecting resistance of mycobacterium tuberculosis to pyrazinamideObjective: To evaluate the reliability and clinical value of Wayne method for the detection of pyrazinamide resistance among MTB strains.Methods: A total of 91 clinical isolates were recruited.Wanye method,BACTEC MGIT960 method,and pnc A gene and its promoter region sequencing were performed as described before.Results: According to the the BACTEC MGIT960 method,the sensitivity,specificity and accuracy of the Wanye method was79.3%(23/29),83.9%(52/62)and 82.4%(75/91),respectively.According to the the pnc A gene sequencing method,the sensitivity,specificity and accuracy of the Wanye method was 72.5%(29/40)、92.2%(47/51)and 83.50%(76/91),respectively.Conclusion: The relability of Wayne method to detect PZA resistance is low,whereas the method needs high volume of bacteria,the outcome is judged by naked eyes,and the procedure is tediouys,which makes it not feasible for clinical use.