Upward trends in new, rifampicin-resistant and concurrent extrapulmonary tuberculosis cases in northern Guizhou Province of China

Similar to global trends, the incidence rate of tuberculosis (TB) in China declined from 2000 to 2018. In this study, we aimed to evaluate TB trends in northern Guizhou Province and identify risk factors associated with rifampicin-resistant (RR) and concurrent extrapulmonary TB (EPTB). We analyzed data of TB patients hospitalized in Affiliated Hospital of Zunyi Medical University from 2011 to 2018, and assessed correlations between demographic characteristics of patients and RR-TB as well as concurrent EPTB. Our results showed that numbers of new, retreated, RR-TB and concurrent EPTB cases increased gradually from 2011 to 2018. Retreated patients had the highest odds of RR-TB but a lower likelihood of concurrent EPTB compared to new patients. Patients between 21 and 40 years of age had a higher likelihood of RR-TB compared to those 20 years and younger. Female patients and patients from Bijie city as well as the Miao ethnic minority had higher odds of concurrent EPTB. In summary, our data demonstrate upward trends in new, rifampicin-resistant and concurrent extrapulmonary TB cases in northern Guizhou Province of China, which should not be overlooked especially during and post the COVID-19 pandemic because TB is a greater long-term global health threat than COVID-19.

www.nature.com/scientificreports/ (10.9%) in Guizhou Province was also higher than the average numbers in China and in the world 12 . Analysis of 8 years of data from four provinces in western China showed that the overall incidence rate of pulmonary TB (PTB) in ethnic minority areas increased gradually from 114.4/100,000 in 2011 to 140.9/100,000 in 2018 13 . Our previous study indicated that the trends in drug-resistant and MDR-TB declined at a major hospital in Guizhou Province from 2008 to 2015 14 .
In order to enhance control strategies and improve treatment outcomes for RR-TB and EPTB, it is important to evaluate TB epidemic characteristics and trends in RR-TB and concurrent EPTB in high TB burden areas of Guizhou Province. The objectives of this study were to extend our previous study by analyzing data collected from 2011 to 2018 about inpatient TB cases at the same hospital; to evaluate the current TB trends and geographic variations in northern Guizhou Province of China; and to identify risk factors associated with RR-TB and concurrent EPTB.

Ethics approval. This observational retrospective study was approved by the Ethics Committee of Affiliated
Hospital of Zunyi Medical University, China. Because diagnostic tests were routinely used in clinical practice and all individual patient information was removed before analysis, the Ethics Committee of Affiliated Hospital of Zunyi Medical University waived the requirement of informed consent from individual patients.

Data sources and procedures. This study was carried out at the Affiliated Hospital of Zunyi Medical
University, which is a tertiary general hospital and one of the major medical centers within the healthcare system of Guizhou Province. This hospital was designated by the provincial government in 2014 as a main part of a regional referral system specialized in the treatment of MDR-TB patients diagnosed at the hospital and those referred from primary/secondary level facilities in northern Guizhou Province, which consists of three prefectural cities (Bijie, Tongren, and Zunyi) (Fig. 1B).
Demographic and clinical data of patients admitted to the TB Division of the hospital were collected and recorded from January 1, 2011 to December 31, 2018. The collected data were stored in the data center of our hospital, which included patient information (gender, age, ethnicity, and address), treatment history, PTB, concurrent EPTB, and drug susceptibility testing (DST) results. To ensure the data used in this study were anonymous, any information related to patient identity was filtered under the supervision of the data center manager.  Clinical isolates of M. tuberculosis (M. tb) were collected from sputum samples of TB or suspected TB patients as part of routine hospital laboratory procedures, and only one isolate per patient was collected to avoid duplication. Processing and inoculation of collected sputum specimens were performed at the Laboratory of Respiratory Medicine of the hospital by following the procedures recommended by WHO. The Laboratory was certified by the China CDC to conduct DST on TB isolates, and DST procedures were described previously 14,15 . Four methods for M. tb detection and two methods for rifampicin (RIF) susceptibility testing were conducted in this study. GeneXpert MTB/RIF (Xpert) (Cepheid Inc.; Sunnyvale, CA, USA) and Loopamp MTBC Detection Kit (TB-LAMP) (Eiken Chemical Co.; Tokyo, Japan) were used in accordance with the manufacturer's instructions. All methods used in this study were performed in accordance with the relevant guidelines and regulations.
Statistical analysis. We used Stata version 16.0 (StataCorp, College Station, Texas, USA) for statistical analyses of linear regression, univariate and multivariate logistic regression, and confidence intervals (CI). The trends of new, retreated, PTB, PTB concurrent with EPTB, rifampicin-susceptible and -resistant TB cases were analyzed by chi-square test. We created maps and annotated geographic and clinical data in those maps using GeoDa software (Version 1.14) downloaded from the GeoDa website (https:// geoda center. github. io). The map data of Guizhou Province of China and cities in Guizhou were from the GADM website (https:// gadm. org/), while the map of administrative units in Zunyi was modified according to the current administrative divisions.

Results
Among 11,610 TB cases collected from 2011 to 2018, 150 (1.3%) EPTB-only cases were excluded from this study because they were not common and the risk of transmission by EPTB-only patients was much lower than patients with PTB and PTB concurrent with EPTB. We analyzed 11,460 PTB cases, of which 98.9% (11,334) were from Guizhou Province, and 1.0% (116) from other provinces of China. The geographical locations of Guizhou Province and its prefectural cities were shown in Fig. 1. Among TB cases from Guizhou, 10,993 (97%) were from northern Guizhou, 8537 from Zunyi, 1325 from Tongren, and 1131 from Bijie (Fig. 1B, Table 1).
Overall, 7012 male (61.2%) and 4448 female (38.8%) TB patients were diagnosed, and 21.2% of male patients and 17.9% of female patients were retreated. Female patients had lower odds of being a retreated TB case (Odds ratio (OR) of 0.81) but higher odds of being a concurrent EPTB case (OR 1.28) than male patients. The population of Guizhou Province is composed of Han ethnicity, which makes up the majority of the population, and many ethnic minorities. In this study, the Han majority accounted for 88.9% of TB patients, while 17 ethnic minorities accounted for 11.1% of TB patients ( Table 1). The top three ethnic minority groups were Tujia, Miao, and Kelao.
The majority of TB patients were over 18 years of age (94%), and 6% of them were between 11 and 17 (Median 48 years, range 11-99). Compared to those 20 years and younger, patients in the 41-60 age group had the highest odds of having retreated TB (OR 4.07) and the lowest odds of having concurrent EPTB (OR 0.59). Patients from the Miao ethnic group had the highest likelihood of having EPTB (OR 1.49, P < 0.0001) in comparison with the Han majority. Additionally, patients from Bijie had the highest odds of having EPTB (OR 1.49, P < 0.0001) compared to those from Zunyi (Table 1).
We have used Xpert assay to simultaneously detect M. tuberculosis complex (MTBC) and RIF resistance since 2016, and have used TB-LAMP to detect MTBC since 2017. At least one of the four detection methods was applied to 98.5% (11,286/11,460) of cases with positive rates of 25.6% for AFB smear, 39.4% for bacterial culture, and 48.4% for Xpert. The percentage of positive PTB cases detected by any of the four methods was 35.4%. During the study period, the proportion of confirmed M. tb cases increased from 21.3% in 2012 to 34.7% in 2016. With the addition of Xpert and TB-LAMP, this proportion increased to over 49% in 2017 and 2018. Overall, 15.7% of tested cases for RIF were RR-TB (Table 2).
We conducted univariate and multivariate logistic regression analyses of risk variables for association with important TB characteristics such as retreated and RR-TB. As shown in Tables 1 and 2, patients from other cities (ORs between 1.29 and 1.49) had higher odds of having concurrent EPTB compared to those from Zunyi, and retreated patients had the highest odds (OR 5.42 and adjusted odds ratio (AOR) 5.17, P < 0.0001) of being RR-TB compared to new patients. Furthermore, patients between 21 and 40 years had a higher likelihood (OR 2.44, P < 0.0001; AOR 1.73, P = 0.021), while patients over 60 years had a lower likelihood of being RR-TB compared to those 20 years of age and younger. Patients with concurrent EPTB had lower odds of being RR-TB (OR 0.65, P = 0.001; AOR 0.73, P = 0.018) compared to PTB patients (Table 2).
To analyze the trends, we compared annual TB cases from 2011 to 2018. During the study period, the number of new TB cases increased 1.84 times, the number of retreated TB cases increased 1.21 times, and the number of concurrent EPTB cases increased 1.71 times (Table 3, Fig. 2). Overall, annual numbers of new, retreated, RR-TB, and concurrent EPTB cases increased from 2011 to 2018 in northern Guizhou Province of China; and the upward trends were statistically significant from 2015 to 2018 (P < 0.05) ( Three past achievements of our department which enhanced the brand awareness of the hospital might have indirectly influenced the fluctuation in percentages of RR-TB and concurrent EPTB cases from 2011 to 2015. First, our department was designated by the provincial government as the "Key Clinical Department of Guizhou Province" in 2011. Second, our department received grants from the "Global Fund" and "National Science and Technology Major Projects" to support patients with MDR/RR-TB to receive standardized diagnosis and treatment between 2011 and 2013. Third, our hospital was designated by the provincial government in 2014 as a specialized hospital for MDR-TB patients. Consequently, more patients with RR-TB, concurrent EPTB, and complex diseases were referred to our hospital from 2011 to 2015. These effects gradually stabilized over the time; therefore, we think that the period from 2015 to 2018 would reflect more practically the current TB trends in northern Guizhou Province of China.  www.nature.com/scientificreports/ The proportion of laboratory confirmed TB cases had increased from 2011 to 2018, especially in recent years with the addition of molecular tests, indicating a continuous improvement in diagnosis of TB. The higher proportion of laboratory confirmed TB cases also provided more isolates for DST, resulting in improved treatment outcomes. The percentage of retreated TB cases is an important indicator of treatment outcomes. Data from the Fifth National TB Epidemiological Survey (2010) showed that percentages of retreated TB cases in both urban and rural areas of western China were higher than those in eastern and central regions of China 20 . However, the percentage of retreated TB cases from this study (20%) was slightly lower than that of China (21%). A steady downward trend in the annual percentage of retreated TB cases from 21.6% (2015) to 18.4% (2018) showed improved treatment success rates in the region and indicated that the increasing number of total TB cases was mainly caused by primary transmission rather than treatment failure.
Retreatment, RR-TB and EPTB are negative factors affecting the treatment outcome and prognosis of patients. Previous treatment history has been a well-known risk factor associated with drug-resistant TB including RR-TB [21][22][23] . Our result that patients between 21 and 40 years of age had higher odds of being RR-TB compared to those 20 years and younger was consistent with past studies 21,24,25 . However, more studies would be needed to verify our result that patients over 60 years had the lowest likelihood of being RR-TB compared to those in other age groups, since the difference was not statistically significant.
Previous studies considered ≥ 45 years of age, female gender, HIV-positive, and the end-stage rental disease as risk factors for EPTB [26][27][28] . However, our study showed that female gender and ≤ 20 years of age were more likely to have concurrent EPTB, which were consistent with reports from high-burden countries such as Nepal, China and Pakistan [29][30][31] . Our result that patients with concurrent EPTB had lower odds of being RR-TB was consistent with past studies 32 . One possible explanation is that most concurrent EPTB cases in our study were new cases which were less likely to be associated with RR-TB. Among different ethnic groups, patients from the Miao ethnic group had the highest likelihood of having EPTB in comparison with the Han majority, which might be better explained by geographic, socioeconomic, and systemic factors. Our study also showed that retreated and RR-TB cases were correlated with each other but they were less likely to be associated with concurrent EPTB.
Our study has a few potential limitations. First, hospitalized patients tend to be more serious and patients attending the University Hospital may be more prestigious or wealthy than outpatients, therefore, actual proportions of retreated, RR-TB and concurrent EPTB cases in northern Guizhou Province might be lower than what we reported here, but trends in the region should remain the same. Similarly, Beijing Chest Hospital reported the RR-TB proportion of 30.5% in 2012 33 , which was higher than the national average. Second, due to the limited scope of the assay, the GeneXpert MTB/RIF cannot detect uncommon mutations in the rpoB gene such as the Ile572Phe (Ile491Phe in the M. tb numbering system) mutation, especially in geographical locations where this mutation is frequent 34,35 . Therefore, the actual proportion of RR-TB could be slightly higher than what we observed in this study. Third, the findings of this study were based only on the data from one major hospital in northern Guizhou province and should not be used to represent the entire Guizhou province because of limitations related to access to the data for the entire province. www.nature.com/scientificreports/ In conclusion, our findings show upward trends in new, RR-TB, and concurrent EPTB cases in northern Guizhou Province of China, indicating that trends in RR-TB and concurrent EPTB could go up or down sporadically in some regions of high TB burden countries, even though overall trends in TB cases continued to decline for the past 20 years in most of the 30 high TB burden countries. To achieve the 2030 targets of the End TB Strategy to reduce the number of TB deaths by 90% and the TB incidence rate by 80% in comparison with levels in 2015, it will be important to enhance TB control programs in regions of the high TB burden countries with upward trends in RR-TB and concurrent EPTB cases. The TB experts who work with governments and international organizations should make specific recommendations to help high TB burden countries to decide which policies they should implement according to their own missions and capabilities.