Persistently high prevalence of primary resistance and multidrug resistance of tuberculosis in Heilongjiang Province, China

All the data we collected including patient medical records are available in Harbin Chest Hospital. A total of 1427 isolates from 1427 patients, who were diagnosed with TB at the Harbin Chest Hospital from May 2007 and July 2012 and from various regions of Heilongjiang Province, were included. The patients were HIV-1 negative. The patient information of gender, age, TB treatment history, and the presence of cavity on chest radiographs was from hospital medical records. Ethical clearance and approval for this study was provided by the Institutional Research Board of the University of Harbin Medical University (Ethics Reference No.: HMUIRB20160001).

Mycobacterial cultures were obtained from clinical specimens after incubation in a BACTEC Mycobacterium Growth Indicator Tube (MGIT) 960 Automated System (BD Diagnostic Systems, Franklin Lakes, NJ, USA). Primary identification was performed using the Ziehl-Neelsen staining method and microscopy. The tests of inhibition by P-nitrobenzoic acid and 2-Thiophenecarboxylic acid hydrazide were used to differentiate M. tuberculosis from other Mycobacterium spp. DST to first-line drugs was performed using MGIT 960 SIRE Kit. Strict controls including growth control (M. tuberculosis H37Rv growth control in drug-free tube) and the negative control (H37Rv in the presence of each drug) were used according to the instruction of MGIT 960 SIRE Kit.

Resistance of some MDR isolates from 2009 to 2012 to FQs was examined in the Hospital as clinical departments suggested. The susceptibility of the MDR strains isolated in 2009 (n = 52, recovered from the frozen stock) to FQs including levofloxacin (Sigma), sparfloxacin (Sigma), moxifloxacin (Hubei Saibo Pharmaceutical company, Wuhan, China), gemifloxacin (Hubei Saibo Pharmaceutical company, Wuhan, China), and gatifloxacin (National Institutes for Food and Drug Control, Beijing, China) was examined by minimun inhibition concentrations (MIC) assay. The test was performed using the standard microdilution method as described previously [17]. Initial concentration was 16 mg/L for both levofloxacin and sparfloxacin, and 8 mg/L for moxifloxacin, gemifloxacin, and gatifloxacin. Seven serial two-fold dilutions of each drug in Middlebrook 7H9 were performed [18‐20]. Each well was inoculated with a 0.03 McFarland mycobacterial suspension (final inoculums was 2.85 × 10⁵ CFU/mL). Two wells without drugs were inoculated with H37Rv as a growth control. All peripheral wells of the plates were filled with sterile distilled water. The plates were incubated at 37 °C for 16 to 18 days. For each drug, the lowest concentration that displayed no visible turbidity was defined as the MIC. Three independent assays were performed.

The susceptibility of MDR isolates from 2010 to 2012 to levofloxacin was determined by using absolute concentration method on solid Lowenstein-Jensen medium (Hangzhou Genesis Biodetection & Biocontrol Ltd) upon National Guideline. The concentration of 2 mg/L was set as the breakpoint [21].

Bacterial DNA was extracted after the inactivation of mycobacterial isolates in 70 % ethanol for 2 h [22]. DNA was extracted using lysozyme and the phenol-chloroform method [23]. Molecular identification of M. tuberculosis with the PCR amplifications of the genes including 16S rRNA, Rv0577, Rv2073c, and Rv3120 was performed as previously described [24, 25]. M. tuberculosis strains have positive amplifications of the four genes. M. microti and M. tuberculosis H37Rv was used as reference strains.

To identify the Beijing genotype of the isolates, RD105 deletion PCR was performed as described previously [24].

The odds ratio was used to evaluate the univariate and multivariate risk factors associated with MDR-TB and drug resistant- (not MDR) TB. The following variables were included in the analysis: the patient’s gender, age, TB treatment history, presence of a cavity on chest radiograph, hospitalization time and Beijing genotype. All of the variables were initially included in the model, and the forward method was used to select the final variables. The statistical interaction between relevant variables was assessed. All statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA).

Of the 1427 patients with pulmonary tuberculosis identified from May 2007 through July 2012, 918 (64.3 %) and 509 (35.7 %) were new and re-treated cases, respectively (Table 1). The medical records were available for all cases. Chest radiographs indicating the presence or absence of cavity were available for 1254 cases. Three hundred isolates from May 2007 through September 2009 were re-confirmed by PCR-based identification and were further examined to identify the Beijing family genotype and phylogeny based on the VNTR genotypes. Fifty two re-cultured available MDR M. tuberculosis isolates from 2009 were examined for sensitivity to fluoroquinolones (MIC). The susceptibility of MDR isolates from 2010 to 2012 to levofloxacin was available from routine clinical drug resistance examinations in hospital.

Overall, the average resistance rate of TB to any first-line drugs from 2007 to 2012 was 57.0 % (Table 1). The drug resistance rate among new case and re-treated case were 48.7 and 72.1 % respectively. The MDR-TB rate was 22.8 %. Of the MDR cases, 13.6 and 39.3 % were new and re-treated cases, respectively. The highest prevalence of resistance to the first-line drugs and MDR from either the new or retreated cases occurred in 2009 and the lowest occurred in 2011 (Table 1).

There was also a high prevalence of primary resistance (Table 1). Of the cases resistant to any first-line drug and MDR-TB cases, approximately 55 and 39 %, respectively, were new cases. From 2007 to 2012, the yearly resistance to any first-line drug among new cases, was 49.2, 51.8, 60.2, 51.7, 33.8, and 39.5 %, respectively. The MDR rates were 15.9, 16.5, 17.0, 12.6, 8.1, and 8.9 %, respectively. Notably, 5.0 % of the pan-resistant cases (resistant to all 4 first-line drugs) were detected in the new cases.

Importantly, the resistance of the re-treated cases was significant (Table 1). Of the cases resistant to any of the first-line drugs and MDR-TB cases, approximately 45.1 and 61.5 %, respectively, were re-treated cases. From 2007 to 2012, the resistance rates of the re-treated cases to any first-line drug were 66.7, 73.8, 82.7, 77.0, 62.3, and 64.4 %, respectively. The annual rates of MDR were 33.3, 41.5, 50.0, 34.4, 31.9, and 37 %, respectively. Of the re-treated cases, 18.5 % were pan-resistant.

Because the resistance rate to the first-line drugs in 2009 was the highest, resistance to fluoroquinolones of some MDR isolates (MIC) was conducted upon suggestion of clinical departments in strengthening the efficacy of treatment (Table 2). The critical concentration for levofloxacin is 2.0 mg/L, as per the World Health Organization, and was used to categorize the isolates as susceptible or non-susceptible. Because there are no recommended critical concentrations for sparfloxacin, moxifloxacin, gatifloxacin and gemifloxacin, we further analyzed the proportion of isolates with MICs equal to and higher than 2 mg/L, based on the serum concentrations of the drugs [20], and they were defined as non-sensitive to a drug.

Of the MDR isolates, 36.5, 26.9, 38.4, and 40.4 % were not susceptible to sparfloxacin, moxifloxacin, gemifloxacin and gatifloxacin, respectively. We also found that there were high proportions of MDR M. tuberculosis isolates resistant to the fluoroquinolones (Table 3). Approximately 21 % of the isolates were not susceptible to levofloxacin and moxifloxacin. The resistance rates to the 3rd and 4th generation of fluoroquinolones were 15.4–17.3 % and 13.5–15.4 %, respectively. Notably, 13.5 % of the isolates were not susceptible to 5 fluoroquinolones.

The risk factors associated with drug resistance were analyzed (Table 4). We found that an age of 35 to 54 (OR 2.5, 95 % CI 1.8–3.4), re-treatment history (OR 5.3, 95 % CI 4.0–7.1), the presence of cavity (OR 1.8, 95 % CI 1.3–2.5), shorter hospitalization (OR 6.4, 95 % CI 4.8–8.7), and infection with the Beijing genotype (OR 4.3, 95 % CI 1.2–15.1) were high-risk factors for the development of MDR-TB based on the univariate analysis. The multivariate analysis further confirmed these findings, except that the Beijing genotype was not included in the final logistic-regression models [26].

Furthermore, MDR-TB cases had a higher frequency (61.5 %) of re-treatment compared to the drug resistant-TB cases (34.2 %). Of the MDR cases, 60.3 % had non-standard hospitalization history compared to 19 % of the drug resistant-TB cases.

Overall, the proportion of cases with cavity, aged 35 to 54, and re-treatment history were 64.8, 41.1, and 35.7 %, respectively, in the TB patients from the study area (Table 4). In this region, the Beijing genotype prevalence was 89.7 %. However, the proportion of cases with non-standard hospitalization was 28.5 %.