Analysis of pyrazinamide resistance of multidrug-resistant Mycobacterium tuberculosis in Henan Province and its relationship with the mutation of drug resistance-related gene pncA / 中华传染病杂志

Objective:To evaluate the performance of the sequencing method of pncA mutations in detecting pyrazinamide (PZA)resistance in multidrug-resistant tuberculosis (MDR-TB) patients in Henan Province. Methods:Sputum samples of 152 MDR-TB patients were collected from 10 drug-resistance surveillance areas in Henan Province from January to December 2018. Questionnaire survey was conducted to collect the information, such as age, gender, treatment history and sputum culture results. The questionnaire and strain samples were sent to Henan Provincial Center for Disease Control and Prevention for further study. PZA susceptibility test was performed using the liquid medium for Mycobacterium tuberculosis. The mutations of pncA were detected by polymerase chain reaction (PCR)and sequencing, then compared with the sequence of standard strain H37Rv. The association between PZA resistant phenotype and treatment outcomes was also investigated. Chi square test and independent sample t test were used for statistical analysis. Results:Among 152 MDR-TB isolates, 105 showed phenotypically PZA resistant. The proportion of PZA resistance in the isolates with isoniazid, rifampicin, ethambutol and streptomycin resistance, pre-extensively drug-resistant and extensively drug-resistant (XDR) were 80.39%(82/102), 81.13%(43/53) and 92.59%(25/27), respectively. One hundred and two isolates had mutations in the pncA gene. Based on the results of the phenotypic drug sensitivity test, the sensitivity of pncA gene mutation detection for PZA resistance was 89.52%(95% confidence interval ( CI) 81.64%-94.39%), and the specificity was 89.36%(95% CI 76.10%-96.01%). These MDR-TB isolates harbored 100 different mutation patterns in the pncA gene, including 80 point mutations and 20 indel mutations, and 13 isolates harbored multiple mutations. Seven strains had mutation in the promoter of pncA, including -7G insertion, -11T to C and -12A to G. The relative expression levels of pncA mRNA in the three groups were 0.21±0.05, 0.31±0.08 and 0.33±0.03, respectively, which were all lower than that of H37Rv(1.00). The differences were statistically significant ( t=4.57, 2.43 and 3.65, respectively, all P<0.05). The difference of the sputum negative conversion rates between patients with PZA-resistant isolates and those with PZA susceptible isolates was statistically significant at different time periods after treatment ( χ2=10.01, P=0.02). The negative conversion rate of PZA-resistant patients at the end of six months of treatment was 1.08%(1/93), and that of PZA-sensitive patients was 7.14%(3/42). Conclusions:The PZA resistance in MDR-TB isolates is associated with pncA mutations, which are scatered and diversified. The sputum negative conversion time of PZA-resistant patients is prolonged.

Comparison of Phenotypic and Genotypic methods for Pyrazinamide susceptibility testing.

Objectives: To evaluate Pyrazinamide (PZA) susceptibility results obtained by phenotypic MGIT 960 TB system against enzymatic Pyrazinamidase assay and genotypic pncA gene sequencing. To find the prevalence of infections caused by M. bovis in PZA resistant M. tuberculosis complex isolates. Methods: 33 consecutive PZA resistant and 30 consecutive PZA susceptible isolates reported for PZA susceptibility testing by MGIT 960 TB system were included in this study. Presence of active pyrazinamidase enzyme was sought by using the Wayne assay. The pncA gene was amplified by PCR and then sequenced to screen mutations. All the PZA resistant isolates were further spoligotyped to identify M. bovis, if present. Results: Of 33 PZA resistant strains by MGIT 960, 31 were Wayne assay negative and two were positive. Of the 30 susceptible PZA strains six were Wayne assay negative reporting false resistance. PncA gene sequencing revealed that 32 of the 33 MGIT PZA resistant isolates had diverse nucleotide changes scattered throughout the pncA gene (one isolate did not show any mutation). Of the 30 phenotypically susceptible isolates, 21 were wild types whilst nine isolates showed the presence of a silent mutation C-T at codon 195. Fifteen mutations found in this study has not been described earlier. Not a single isolate of M. bovis was detected among PZA resistant M. tuberculosis complex isolates. Conclusion: MGIT 960 showed better concordance with sequencing results in comparison with Wayne assay. In present study, a high proportion (85%) of MDR-TB isolates from patients receiving anti-TB treatment were found to be resistant to PZA.

Relationship of oncogene PCNA, nm23 expression in breast cancer and its prognostic significance / 中国医师进修杂志

Objective To investigate the relationship between PCNA and nm23 expression in breast cancer tissue and its prognosis. Methods Detected the expression of PCNA and nm23 of 278 cases of breast cancer by immunohistochemical technique S-P and analyzed it combined with the clinicopathology. Results PCNA positive rate was 37.4%(104/278),nm23 positive rate was 59.7%(166/278).PCNA and nm23 genes expression had no obvious correlation with the breast cancer pathological type,patients age and clinical stage (P > 0.05). PCNA expression had the positive correlation with the axillary lymph nodes meta-sitasis but negative to 5-year survival rates(P < 0.01 ),nm23 expression had the negative correlation with the axillary lymph nodes metasitasis but positive to 5-year survival rates(P < 0.01 ). PCNA and nm23 expression had the obvious negative correlation between them(P < 0.01 ). Conclusion Combined detection the expres-sion of PCNA and nm23 in breast cancer tissue will be important reference to forcast and evaluate the prognosis of the breast cancer patients.

Characterization of pncA Mutations of Pyrazinamide-Resistant Mycobacterium tuberculosis in Korea

Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.

Detection of Pyrazinamide Resisitance in Mycobacterium Tuberculosis by Sequencing of pncA Gene / 결핵

BACKGROUND: Examining the biological susceptibility of Mycobacterium tuberculosis to pyrazinamide (PZA) in vitro is very difficult as PZA is inactive under normal culture conditions. The susceptibility test, an enzyme assay for Pzase activity, and a genetic test for pncA gene mutations, were performed in order to predict PZA resistance. METHODS: 28 cultured clinical isolates of Mycobacterium tuberculosis were tested. The biological susceptibility was performed by the absolute concentration method using Lowenstein-Jensen media. The PZase activity was tested by means of Wayne's method. A 710-bp region includes the entire open reading frame of pncA was amplified and sequenced. RESULTS: All six strains with positive PZase activity exhibited no pncA mutations with one strain showing a false resistance in the biological susceptibility test. Among the 22 strains with no PZase activity, 21 exhibited showed pncA mutations. In the biological suscaptibility test, 20 strains were resistant, and one was susceptible, and the other failed to test. The mutation types varied with ten missense, one silent and one nonsense mutation 1 slipped-strand mispairing, and 6 frameshift mutations. Three strains had an adenine to guanine mutation at position - 11 upstream of the start codon. CONCLUSION: The mutation at the pncA promotor region is frequent at -11 upstream position. Automatic sequencing of pncA is a useful tool for rapid and accurate detection of PZA resistant M.tuberculosis, and for demonstrating the epidemiological relatedness of the PZA-resistant M.tubersulosis strains.

Changes in midpalatal suture area and adjacent periodontal tissues of individual tooth following rapid palatal expansion in young adult dogs ; Histomorphologic and immunohistochemical study / 대한치과교정학회지

The purpose of this study was to determine the proliferative activity of the osteoblasts and fibroblasts in the midpalatal area and to investigate the adjacent periodontal tissues of individual tooth following rapid expansion of the palate. Ten young adult dogs, aged approximately ten months, were used in the experiment. The experimental design was consisted of 1 week expansion group(Group E1, 3 dogs), 2 week expansion group(Group E2, 3 dogs), 2 week expansion and 2 week retention group(Group E3, dogs), and control group(Group C, 1 dog). For each group, expansion screw was activated one time per day(1/4 turn;90degrees) following Hyrax-screw application. The experimental animals in each group were sacrificed at 1, 2 and 4 weeks following palatal expansion. Maxillary tissue blocks wee obtained and prepared for the histomorphologic and immunohistochemical studies. Light microscope, polarizing microscope, and soft X-ray apparatus were used in this study, and following results were obtained. 1. In polarizing microscopic study, the expansion group(E1 & E2) showed blue color representing bone resorption and new bone formation in midpalatal suture area. E3 groups showed less color compared to the E1 and E2 group. But yellow color increased by calcification in the E3 groups. 2. Immunohistochemical study revealed that positive responses of the osteoblasts to PCNA and undifferentiated fibroblasts to EGF in E1 group were somewhat increased. Positive response to PCNA and EFG were increased in fibroblasts and the osteoblasts forming new bone in E2 group. In E3 group, the positive response cell concentrated the periphery of edge of palatal process in both PCNA and EGF. 3. Throughout the expansion period(E1 & E2), light microscopic study showed the edges of the extensive resorption new palatal processes, indication bone remodeling within the suture. E3 group exhibited less remodeling of midpalatal suture area. E2 group and E3 group showed cementum formation and resorption at the apex of 3rd premolar and 1st molar. E3 group exhibited extensive hyalinized zone on the cervical portion of buccal side of 1st molar. 4. Soft X-ray analysis of E1 group showed hypomineralized defect and microfractures in various parts of the suture areas when compared with control animals. There was no significant difference in the degree of mineralization in the midpalatal suture region between the C and E3 groups. Tooth axis showed tipping of 3rd premolar and 1st molar in the E2 group and E3 group. Based upon these experimental results, it is concluded that the undifferentiated mesenchymal cells always presented in midpalateal suture area following RPE. Differentiated osteoblasts and fibroblasts possess proliferating cellular activity until the 2 week retention period. The posterior teeth area tend to tip buccally as RPE force applied. Retention group exhibited irreversible response with severe hyalinized on the buccal surface of the first molar.