Time-trend analysis of tuberculosis diagnosis in Shenzhen, China between 2011 and 2020.

Objective: To describe the trend of tuberculosis (TB) diagnosis in the migrant city Shenzhen, China, and analyze the risk factors of diagnosis delays. Methods: Demographic and clinical information of TB patients from 2011 to 2020 in Shenzhen were extracted. A bundle of measures to enhance TB diagnosis had been implemented since late 2017. We calculated the proportions of patients who underwent a patient delay (>30 days from syndrome onset to first care-seeking) or a hospital delay (>4 days from first care-seeking to TB diagnosis). Multivariable logistic regression was used to analyze the risk factors of diagnosis delays. Results: During the study period, 43,846 patients with active pulmonary TB were diagnosed and registered in Shenzhen. On average, the bacteriological positivity rate of the patients was 54.9%, and this increased from 38.6% in 2017 to 74.2% in 2020. Overall, 30.3 and 31.1% of patients had a patient delay or a hospital delay, respectively. Molecular testing significantly increased bacteriological positivity and decreased the risk of hospital delay. People >35 years old, the unemployed, and residents had a higher risk of delays in both patient care-seeking and hospital diagnosis than younger people, workers, or migrants. Compared with passive case-finding, active case-finding significantly decreased the risk of patient delay by 5.47 (4.85-6.19) times. Conclusion: The bacteriological positivity rate of TB patients in Shenzhen increased significantly but the diagnosis delays were still serious, which may need more attention when active case-finding in risk populations and optimization of molecular testing.

[Drug sensitivity analysis of Mycobacterium chelonae and Mycobacterium abscessus and evaluation of Etest for susceptibility testing].

OBJECTIVE: To study the drug resistance profile of Mycobacterium(M.) chelonae and M.abscessus and to evaluate the clinical application of Etest(epsilometer test) for susceptibility testing. METHODS: Twenty clinical isolates of M.abscessus and 16 clinical isolates of M.chelonae from clinical specimens were collected.Strain identification was carried out by GenoType Mycobacterium CM assay(Hain Lifescience, Germany). The accuracy was evaluated by comparing Etest results to those obtained by broth microdilution. Thirty-six isolates were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, sulfamethoxazole and tobramycin. The agreement among MICs and interpretive category was evaluated. Chi-squared test was used to compare observed frequency of each of the 2 examples. RESULTS: All of the isolates(36/36) were sensitive to amikacin and cefoxitin, and only 1 isolate(1/36) was resistant to clarithromycin, but more isolates(29/36) were resistant to ciprofloxacin, doxycycline, imipenem and sulfamethoxazole.For M.chelonae, only 2/16 were resistant to linezolid, and 7/16 resistant to tobramycin.For M.abscessus, more than 12/20 were resistant to linezolid and 16/20 resistant to tobramycin. The agreement between broth microdilution MICs and Etest MICs for 9 drugs was 149/324.With amikacin, clarithromycin, doxycycline and imipenem, the agreement for interpretive category was excellent(35/36), followed by sulfamethoxazole(34/36), which corresponded to rarely very major error of 2/36.With ciprofloxacin and tobramycin, agreement for interpretive category was 31/36 and 26/36.With cefoxitin and linezolid, the agreement of Etest MICs was the lowest(14/36), resulting in the resistant category. CONCLUSIONS: Isolates of M.chelonae and M.abscessus exhibit far more susceptibility to amikacin, cefoxitin and clarithromycin than any other antimicrobial agents.Linezolid and tobramycin showed sensitivity to some isolates of M.chelonae.It is suitable for the Etest method as a simple reliable method for the drug susceptibility of amikacin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and tobramycin except to cefoxitin and linezolid. The Etest method of determining sulfamethoxazole susceptibility should be careful.

[Detection of pncA mutation associated with pyrazinamide resistance in Mycobacterium tuberculosis by high-resolution melting cure analysis].

OBJECTIVE: To evaluate the application of a high-resolution melting (HRM) curve analysis for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis (M. tuberculosis). METHODS: Clinical isolates of M. tuberculosis were collected from Shenzhen from 2007 - 2009 and pncA mutations were characterized by sequence analysis. HRM analysis method was established and used to determine pncA gene mutation in 49 pncA mutant isolates and 78 wild type isolates. BCATEC MGIT 960 was used as the standard method to evaluate application of the HRM curve. RESULTS: Among 49 pncA mutant isolates, 48 isolates were characterized as mutant and 1 isolate with double point mutations misclassified as wild type by using HRM detection. In 78 wild type isolates, HRM found 6 mutant isolates. Using the BCATEC MGIT 960 to detect pyrazinamide drug susceptibility, 62 isolates were found resistant and 65 were susceptible. Among the 62 resistant strains by HRM results 53 strains were resistant, and among the 65 susceptible stains by HRM results 64 were susceptible. Compared to the results of BACTEC MGIT 960, the sensitivity and specificity of HRM were 85.5% (53/62) and 98.5% (64/65), respectively. The Youden index was found to be 0.840. CONCLUSIONS: The results showed a good correlation between HRM curve analysis and BACTEC MGIT 960. HRM curve analysis is a rapid and accurate method that can be used as a screening test for identification of resistance to pyrazinamide.

[Characteristics of pncA gene in multidrug-resistant Mycobacterium tuberculosis isolates and its correlation with drug resistance to pyrazinamide].

OBJECTIVE: To understand the characteristics of pncA gene in multidrug-resistant Mycobacterium tuberculosis isolates and its correlation with drug resistance to pyrazinamide. METHODS: A total of 127 clinical isolates of multidrug-resistant mycobacterium tuberculosis were collected from Shenzhen from year 2007 to 2009. PZA susceptibility was determined by the BACTEC MGIT 960 PZA method. Pyrazinamidase (PZase) activity testing and pncA gene sequencing were performed in all the isolates. The type and frequency of mutations in pncA were determined. Correlation analysis among PZA susceptibility and PZase activity, pncA mutation was performed. RESULTS: Among the 127 isolates, 62 isolates (48.8%) were found resistance to PZA. Among the 62 PZA resistant isolates, 45 isolates which had various pncA mutations were negative for PZase. Mutation rate was 77.4% (48/62) in total PZA resistance isolates. Different types of 48 resistant isolates were identified in the pncA gene, including base substitution (33 isolates), frame shift mutation (12 isolates) and codon mutation (3 isolates). No mutations except one isolate (N11D) existed in all PZA-susceptibility isolates which were positive for PZase. A total of 5 mutations which have not been described previously were found as follows: H57P, P62Q, G108R, D110Y and G162V. The correlation among the PZA susceptibility and the PZase activity (r = 0.895, P < 0.05), the pncA mutation (r = 0.779, P < 0.05) were significant in 127 multidrug-resistant isolates. CONCLUSION: A high diversity of pncA gene mutation was found among PZA resistant strains of MTB. This study revealed five new mutations of the pncA gene that were not previously described, which scattered in the hot-spot regions located in the metal coordination site and active site of the enzyme. Mutations had a high correlation with the PZA resistance.