Study On The Detection Rate Of Tuberculosis And The Mutation Of PncA And RpsA Genes Related To Pyrazinamide Resistance By Magnetic Beads Enrichment Method

Background:A review of the scientific literature concluded that indirect smear can improve detection of TB in sputum compared to direct smear. However, the operation iscomplicated and need centrifuge, few laboratories have access to centrifugation in order to perform indirect smear. This study investigated whether an alternative method of magnetic bead concentration could enhance diagnosis of TB in laboratories which only perform direct smear microscopy.Methods:One hundred and twenty-nine PTB patients*sputa from Beijing Chest Hospital were enrolled into this study from Jun.2011to Oct.2011.Every sputum have been dealed with by beads(TB-BEADS,The British Microsens Biotech Corporation), then smear sputa were conducted with Ziehl-Neelsen acid-fast staining/optical microscopic examination, and fluorescent staining/Light emitting diode fluorescent microscope to detect tuberculosis(LED fluorescence microscopy), at the same time, direct smear of sputa were conducted with Ziehl-Neelsen acid-fast staining and fluorescent staining to detect tuberculosis, Lowenstein-Jensen culture and Bactec culture as control. Then compare and contrast the above methods. The χ2test was used for comparison.Results:①The Ziehsolid L-J culture as the standard, direct smear had a sensitivity of45%(29/64) by l-Neelsen (ZN) and52%(3364) by auramine. a specificity of95%(62/64) and95%(6264):After TB-Bead extraction, indirect smear had a sensitivity of67%(4364) by Ziehl-Neelsen (ZN) and73%)(47/64) by auramine,a specificity of85%(55’64) and83%(5464).②The liquid MG1T960culture as the standard, direct smear had a sensitivity of43%(3274Ziehl-) by Neelsen (ZN) and49%(36/74) by auramine. a specificity of100%(55/55) and100%(5555):After TB-Bead extraction, indirect smear had a sensitivity of67%(43/64) by Ziehl-Neelsen (ZN) and73%(47/64) by auramine, a specificity of98%(54/55) and95%(52/55).③Combined the two methods as the standard, direct smear had a sensitivity of40%(32/80) by Ziehl-Neelsen (ZN) and45%(36/80) by auramine, a specificity of100%(49/49) and100%(49/49); After TB-Bead extraction, indirect smear had a sensitivity of65%(52/80) and70%(56/80), a specificity of100%(49/49) and98%(48/49).Conclusion:using the same smear method, the smear positive rate of LED fluorescence microscopy is better than that of optical microscope examination;Magnetic bead concentration of mycobacteria from sputum led to a significant improvement (p<0.05) in the sensitivity of microscopy compared with direct smear. Objective:To know mutation of genes pncA and rpsA in the MDR-TB clinical isolates, and analyze the relationship between these mutations and the resistant to pyrazinamide (PZA).Methods:Collected142cases of clinical isolates of MDR-TB, determined these strains’sensitivity of pyrazinamide using the PZA resistance kit(BDcompany.USA) and detected mutations of genes pncA and rpsA by the polymerase chain reaction (PCR).Compared the results with the corresponding sequence of TB H37Rv strain,Combined the corresponding mutation of amino acid sequence, and analysized the gene mutationResults:In the142strains of MDR-TB clinical isolates. PZA resistant strains accounted for43%(61/142). PZA sensitive strains accounted for57%(81/142).The sequencing results of genes pncA and rpsA in142cases of MDR-TB in clinical isolates were compared with H37Rv ATCC. combined with statistical mutation in amino acid sequence corresponding to site. The pncA gene analysis showed:In the61strains of PZA resistant strains,47strains of mutation, the mutation rate of77%(47/61); In the81strains of PZA sensitive strains.27strains of mutation, the mutation rate of33%(27/81). The rpsA gene analysis showed:In the61strains of PZA resistant strains,25strains of mutation, the mutation rate of41%(25’61):In the81strains of PZA sensitive strains.19strains of mutation, the mutation rate of11%(19/81). Mutations in47strains of drug-reistant strains of PZA pncA gene, point mutation accounted for72%(34,47). bases insert accounted for11%(547). base deletion accounted for15%(7/47). composite mutation accounted for2%(1/47):In 27strains of PZA sensitive mutant strain pncA gene, point mutation ratio96%(26/27), base deletion accounted for4%(1/27). Mutations in25strains of drug-resistant strains of PZA rpsA gene, point mutation accounted for52%(13/25), bases insert accounted for12%(3/25), base deletion accounted for24%(6/25), composite mutation accounted for12%(3/25); In19strains of PZA sensitive mutant strain rpsA gene, point mutation ratio79%(15/19), base deletion accounted for16%(3/19), composite mutation accounted for5%(1/19).Conclusions:In the MDR-TB strains from Beijing Chest Hospital,pyrazinamide resistance and pncA gene mutation were highly correlated with the pncA gene mutation, and lowly correlated with the rpsA gene mutation.