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

Background The spread of multidrug-resistant tuberculosis (MDR-TB) Mycobacterium tuberculosis (M. tuberculosis) strains has been a big challenge to the TB control and prevention in China. Knowledge about patterns of drug resistance in TB high-burden areas of China is crucial to develop appropriate control strategies. We conducted a comprehensive investigation of the resistance pattern of M. tuberculosis in Heilongjiang Province. Methods 1427 M. tuberculosis clinical strains were isolated from pulmonary TB patients hospitalized between 2007 and 2012. The susceptibility of the isolates to the first-line anti-TB drugs and the resistance of MDR M. tuberculosis to fluoroquinolones were examined. We also performed a statistical analysis to identify the correlated risk factors for high burden of MDR-TB. Results The global resistance rates of 2007–2012 to the first-line drugs and MDR were 57.0 and 22.8 %, respectively. Notably, the primary MDR-TB and pan-resistance rates were as high as 13.6 and 5.0 %, respectively. Of MDR M. tuberculosis isolates (2009), approximately 13 % were not susceptible to any of the fluoroquinolones tested. Being age of 35 to 54, high re-treatment proportion, the presence of cavity lesion, and high proportion of shorter hospitalization are correlated with the development of MDR-TB. Conclusions The high prevalence of drug resistant, MDR-TB, and fluoroquinolone-resistant MDR-TB is a big concern for TB control. More importantly, in order to control the development of MDR-TB effectively, we need to pay more attention to the primary resistance. Targeting reducing the prevalence of the risk factors may lead to better TB control in China.


Background
The spread of drug-resistant and multidrug-resistant (MDR) tuberculosis (TB) is a severe global health issue. In recent years, both the incidence and prevalence of TB in China have steadily declined [1]. The World Health Organization (WHO) indicates that the MDR rates in China (5.7 % in new cases and 26 % in previously treated cases, respectively) have become lower than in other countries among the 27 high MDR-TB burden countries. However, the rates remain higher than the global average rates of 3.6 and 20 % for new and previously treated cases, respectively. China is ranked second in the number of MDR-TB cases after India because of China's large population. Moreover, the global extensively drugresistant (XDR)-TB prevalence is increasing, with 9.6 % MDR-TB cases in 2012 compared to 5.4 % in 2007. We estimate an increasing XDR-TB prevalence in China, with 8 % from MDR-TB cases, because this rate was higher than the worldwide level in 2007 [1]. The global spread of XDR-TB has led to new challenges for the prevention and control of tuberculosis.
The epidemic trends of drug resistant-TB in different geographic areas of China vary greatly, and recently, the spread of TB in China has been caused by active domestic migration, resulting in a significant public health issue. Heilongjiang Province is located in Northeast China, a region in which the prevalence rates of both TB and drugresistant TB are higher than the average in China [2][3][4]. The rates of any resistance to first-line drugs were 38.9 and 36.2 % for new cases and 70.9 and 67.7 % for retreated cases in Heilongjiang Province in 2002 and 2004, respectively, based on data from population-based drug resistance investigations [2][3][4].
It is important to identify the causes for the high prevalence of drug-resistance TB. The risks associated with drug resistant-and MDR-TB include a history of retreatment using anti-mycobacterial drugs, having cavities, Beijing genotype epidemic, low socio-economic status, age and DOTS (directly observed treatment short course) implementation. However, the factors vary depending on the study [1,[5][6][7][8].
Moreover, we would like to determine whether the prevalence of fluoroquinolone resistant TB in Heilongjiang Province is also high. MDR-TB is a risk factor for the resistance of M. tuberculosis to fluoroquinolones [9,10]. In China, fluoroquinolones are the most frequently used antibiotics for the treatment of a variety of infectious diseases [11][12][13][14]. XDR-TB arises when MDR-TB strains acquire resistance to any fluoroquinolone, and previous treatment using second-line drugs, including fluoroquinolones, is a significant risk factor for XDR-TB [15,16].
In the present study, we analyzed the resistance trends of M. tuberculosis clinical isolates from 2007 to 2012 to first-line drugs and fluoroquinolones and the risk factors in Heilongjiang Province, an area with the highest tuberculosis disease and drug resistance burden in China. Elucidating the locally significant risk factors for the high prevalence of TB is important to control the spread of TB and drug resistant TB in this area and other regions of China.

Mycobacterial specimen and data collection
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 culture and drug susceptibility testing (DST) to first-line drugs
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 drugfree tube) and the negative control (H37Rv in the presence of each drug) were used according to the instruction of MGIT 960 SIRE Kit.

The resistance of MDR M. tuberculosis to fluoroquinolones
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][19][20]. Each well was inoculated with a 0.03 McFarland mycobacterial suspension (final inoculums was 2.85 × 10 5 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].
DNA extraction, PCR-based identification of M. tuberculosis, and Beijing genotype determination 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].

Statistical analysis
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).

Sample and data collection
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.

High prevalence of drug-resistant TB in Heilongjiang Province from 2007 to 2012
Overall, the average resistance rate of TB to any firstline drugs from 2007 to 2012 was 57.0 % ( Table 1). The drug resistance rate among new case and re-treated case Importantly, the resistance of the re-treated cases was significant (Table 1) 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 nonsusceptible. 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.

Factors linked to drug-resistant TB
The risk factors associated with drug resistance were analyzed (  [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-  (Table 4). In this region, the Beijing genotype prevalence was 89.7 %. However, the proportion of cases with nonstandard hospitalization was 28.5 %.
Furthermore, less than 20 % of the TB medical facilities that use second-line drugs for MDR-TB treatment design therapy regimens based on DST [34]. This situation will cause the appearance of additional MDR and XDR M. tuberculosis cases.
In the new cases, high proportions with cavity (approximately 60 %) and short hospitalization (19 %) should also be identified. These patients are released from hospitals after the initial treatment and may be a highly dangerous source of infection for susceptible individuals, causing the spread of both TB and MDR TB. It is important to decrease the proportion of non-standard hospitalization and appropriately treat the cases with cavity to achieve the successful control of primary TB spread. The resistance situation to FQs distributed variously in different TB populations (outpatients and inpatients) and in different areas of China, with a higher resistance rate in hospitalized patients. In addition, the MDR strains would be more likely to resistant to FQs [10,34,38,[43][44][45][46][47].
In recent years, levofloxacin, the most frequently used fluoroquinolone in China, has become the first choice fluoroquinolone for MDR-TB treatment, and moxifloxacin is used as an alternative fluoroquinolone based on China's clinical practice guidelines for TB [26].
Based on the present data, there is a limited choice of fluoroquinolones for the ideal regimen for MDR-TB therapy because approximately 15-17 % and 14-15 % of the MDR-TB cases are not susceptible to the 3 and 4 of fluoroquinolones, respectively. Importantly, at least 13 % of the MDR M. tuberculosis isolates were not susceptible to any of the 5 fluoroquinolones. The high prevalence of drug resistance may become uncontrolled if measures are not effective and may indicate an increase of XDR-TB.
MDR is an independent risk factor associated with resistance to fluoroquinolones [9]. The high rate of MDR-TB in the investigated area (22.8 %) is a related causative reason for the high prevalence of fluoroquinolone resistance. Furthermore, the fluoroquinolones are commonly used antibiotics for clinical departments, and antibiotics abuse has not been well controlled in China [11][12][13][14].
Controlling the resistance to fluoroquinolones may be achieved through the proper management of MDR-TB cases and the proper use of such antibiotics.

Conclusions
The results of our study indicate that there was a high prevalence of drug resistance to the first-line drugs and multidrug resistance (MDR) in Heilongjiang Province, northeastern China. Among MDR TB, more than 10 % was resistant to fluoroquinolones, indicating a severe second line drug resistance. After analysis of the risks of MDR-TB, we should pay more attention to patients' age, re-treatment proportion, cavity lesion, and high proportion of shorter hospitalization to get control tuberculosis more efficiently.