Genomic investigation of bone tuberculosis highlighted the role of subclinical pulmonary tuberculosis in transmission.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) without symptomatic pulmonary involvement has been thought to be non-transmissible, but EPTB with asymptomatic pulmonary tuberculosis (PTB) could transmit tuberculosis (TB). Genomic investigation of Mycobacterium tuberculosis (Mtb) isolates from EPTB may provide insight into its epidemiological role in TB transmission. METHODS: Between January 2017 and May 2020, 107 Mtb isolates were obtained from surgical drainage of bone TB patients at the Beijing Chest Hospital, and 218 Mtb strains were isolated from PTB cases. These 325 Mtb isolates were whole-genome sequenced to reconstruct a phylogenetic tree, identify transmission clusters, and infer transmission links using a Bayesian approach. Possible subclinical PTB in the bone TB patients was investigated with chest imaging by two independent experts. RESULTS: Among 107 bone TB patients, 10 were in genomic clusters (≤12 SNPs). Phylogenetic analysis suggested that three bone TB patients transmitted the infection to secondary cases, supported by epidemiological investigations. Pulmonary imaging of 44 bone TB patients revealed that 79.5 % (35/44) had radiological abnormalities suggestive of subclinical PTB. CONCLUSIONS: This study provides genomic evidence that bone TB patients without clinically diagnosed PTB can be sources of TB transmission, underscoring the importance of screening for subclinical, transmissible PTB among EPTB cases.

The Global Success of Mycobacterium tuberculosis Modern Beijing Family Is Driven by a Few Recently Emerged Strains.

Strains of the Mycobacterium tuberculosis complex (MTBC) Beijing family aroused concern because they were often found in clusters and appeared to be exceptionally transmissible. However, it was later found that strains of the Beijing family were heterogeneous, and the transmission advantage was restricted to sublineage L2.3 or modern Beijing. In this study, we analyzed the previously published genome sequences of 7,896 L2.3 strains from 51 different countries. Using BEAST software to approximate the temporal emergence of L2.3, our calculations suggest that L2.3 initially emerged in northern East Asia during the early 15th century and subsequently diverged into six phylogenetic clades, identified as L2.3.1 through L2.3.6. Using terminal branch length and genomic clustering as proxies for transmissibility, we found that the six clades displayed distinct population dynamics, with the three recently emerged clades (L2.3.4 to L2.3.6) exhibiting significantly higher transmissibility than the older three clades (L2.3.1 to L2.3.3). Of the Beijing family strains isolated outside East Asia, 83.1% belonged to the clades L2.3.4 to L2.3.6, which were also associated with more cross-border transmission. This work reveals the heterogeneity in sublineage L2.3 and demonstrates that the global success of Beijing family strains is driven by the three recently emerged L2.3 clades. IMPORTANCE The recent population dynamics of the global tuberculosis epidemic are heavily shaped by Mycobacterium tuberculosis complex (MTBC) strains with enhanced transmissibility. The infamous Beijing family strain stands out because it has rapidly spread throughout the world. Identifying the strains responsible for the global expansion and tracing their evolution should help to understand the nature of high transmissibility and develop effective strategies to control transmission. In this study, we found that the L2.3 sublineage diversified into six phylogenetic clades (L2.3.1 to L2.3.6) with various transmission characteristics. Clades L2.3.4 to L2.3.6 exhibited significantly higher transmissibility than clades L2.3.1 to L2.3.3, which helps explain why more than 80% of Beijing family strains collected outside East Asia belong to these three clades. We conclude that the global success of L2.3 was not caused by the entire L2.3 sublineage but rather was due to the rapid expansion of L2.3.4 to L2.3.6. Tracking the transmission of L2.3.4 to L2.3.6 strains can help to formulate targeted TB prevention and control.

Mobile APP-assisted family physician program for improving blood pressure outcome in hypertensive patients.

BACKGROUND: This study was aimed to examine the effectiveness of App-assisted self-care in a Beijing community based on intelligent family physician-optimised collaborative model (IFOCM) program.  METHODS: We conducted a survey of 12,050 hypertensive patients between Jan 2014 and Dec 2021. Generalized linear model was used to analyze the covariates that associated with blood pressure (BP) control. Decision tree and random forest algorithm was used to extract the important factors of BP outcome. RESULTS: The study included 5937 patients, mean age 66.2 ± 10.8, with hypertension in the baseline; 3108(52.4) were female. The community management resulted in mean systolic BP and diastolic BP reductions of 4.6 mmHg and 3.8 mmHg at follow-up. There were 3661 (61.6%) hypertension patients with BP control, increasing from 55.0% in 2014 to 75.0% in 2021. After adjusted for covariates, antihypertensive medication adherence, diabetes, and APP-assisted self-care were common predictors associated with BP control in GLM model and machine learning algorithm. CONCLUSION: Community management based on IFOCM program significantly improved BP control in hypertensive patients. APP-assisted self-care would be beneficial for the management of chronic disease.

Comparison of clinical characteristics between COVID-19 and H7N9 fatal cases: An observational study.

Objective: The outbreak of COVID-19 in 2020 is reminiscent of the H7N9 outbreak in 2013, which poses a huge threat to human health. We aim to compare clinical features and survival factors in fatal cases of COVID-19 and H7N9. Methods: Data on confirmed COVID-19 and H7N9 fatal cases identified in mainland China were analyzed to compare demographic characteristics and clinical severity. Survival curves were estimated by the Kaplan-Meier method and compared using log-rank tests and a restricted mean survival time model. A Cox regression model was used to identify survival factors in fatal cases of COVID-19 and H7N9. Results: Similar demographic characteristics were observed in fatal cases of COVID-19 and H7N9. The proportion of fatal cases of H7N9 receiving antibiotics, antiviral drugs, and oxygen treatment was higher than that of COVID-19. The potential protective factors for fatal COVID-19 cases were receiving antibiotics (HR: 0.37, 95% CI: 0.22-0.61), oxygen treatment (HR: 0.66, 95% CI: 0.44-0.99), and corticosteroids (HR: 0.46, 95% CI: 0.35-0.62). In contrast, antiviral drugs (HR: 0.21, 95% CI: 0.08-0.56) and corticosteroids (HR: 0.45, 95% CI: 0.29-0.69) were the protective factors for H7N9 fatal cases. Conclusion: The proportion of males, those having one or more underlying medical condition, and older age was high in COVID-19 and H7N9 fatal cases. Offering antibiotics, oxygen treatment, and corticosteroids to COVID-19 cases extended the survival time. Continued global surveillance remains an essential component of pandemic preparedness.

Transmission of multidrug-resistant tuberculosis in Beijing, China: An epidemiological and genomic analysis.

Background: Understanding multidrug-resistant tuberculosis (MDR-TB) transmission patterns is crucial for controlling the disease. We aimed to identify high-risk populations and geographic settings of MDR-TB transmission. Methods: We conducted a population-based retrospective study of MDR-TB patients in Beijing from 2018 to 2020, and assessed MDR-TB recent transmission using whole-genome sequencing of isolates. Geospatial analysis was conducted with kernel density estimation. We combined TransPhylo software with epidemiological investigation data to construct transmission networks. Logistic regression analysis was utilized to identify risk factors for recent transmission. Results: We included 241 MDR-TB patients, of which 146 (60.58%) were available for genomic analysis. Drug resistance prediction showed that resistance to fluoroquinolones (FQs) was as high as 39.74% among new cases. 36 (24.66%) of the 146 MDR strains were grouped into 12 genome clusters, suggesting recent transmission of MDR strains. 44.82% (13/29) of the clustered patients lived in the same residential community, adjacent residential community or the same street as other cases. The inferred transmission chain found a total of 6 transmission events in 3 clusters; of these, 4 transmission events occurred in residential areas and nearby public places. Logistic regression analysis revealed that being aged 25-34 years-old was a risk factor for recent transmission. Conclusions: The recent transmission of MDR-TB in Beijing is severe, and residential areas are common sites of transmission; high levels of FQs drug resistance suggest that FQs should be used with caution unless resistance can be ruled out by laboratory testing.

Case report: A 9-year systematic treatment failure of a pulmonary tuberculosis patient.

Objective: To explore the reasons of failure in a case of pulmonary tuberculosis (PTB) after 9 years systematic treatment. Methods: We extracted the patients' treatment history, drug susceptibility testing (DST), Computed tomography (CT) images, and sequenced the isolated strains by whole gene sequencing (WGS). Results: Although most results of the phenotypical DSTs were consistent with the genotype DST, the occurrence of gene resistance to amikacin (AMK), capreomycin (CAP), moxifloxacin (MFX) was earlier than the phenotypical DST. Based on the continuously reversed results of phenotypical DSTs, CT images in different stages and WGS, it can be confirmed that the patient was infected with two different strains of Mycobacterium tuberculosis (M.TB). Moreover, severe cavities may be another factor leading to treatment failure. Conclusion: Given the suggestive effect of genotype DST is earlier than the phenotypical DST, so genotype DST can play a better guiding role in patients with MDR-TB. Additionally, for patients who have not been cured for a long time, medication should be more cautious and the role of WGS in drug resistance surveillance should be fully utilized.

Epidemiology of recurrent pulmonary tuberculosis by bacteriological features of 100 million residents in China.

BACKGROUND: Recurrence continues to place significant burden on patients and tuberculosis programmes worldwide, and previous studies have rarely provided analysis in negative recurrence cases. We characterized the epidemiological features of recurrent pulmonary tuberculosis (PTB) patients, estimated its probability associated with different bacteriology results and risk factors. METHODS: Using 2005-2018 provincial surveillance data from Henan, China, where the permanent population approximately were 100 million, we described the epidemiological and bacteriological features of recurrent PTB. The Kaplan-Meier method and Cox proportional hazard models, respectively, were used to estimate probability of recurrent PTB and risk factors. RESULTS: A total of 7143 (1.5%) PTB patients had recurrence, and of 21.1% were bacteriological positive on both laboratory tests (positive-positive), and of 34.9% were negative-negative. Compared with bacteriological negative recurrent PTB at first episodes, the bacteriological positive cases were more male (81.70% vs 72.79%; P < 0.001), higher mortality risk (1.78% vs 0.92%; P = 0.003), lower proportion of cured or completed treatment (82.81% vs 84.97%; P = 0.022), and longer time from onset to end-of-treatment. The probability of recurrence was higher in bacteriological positive cases than those in bacteriological negative cases (0.5% vs 0.4% at 20 months; P < 0.05). CONCLUSIONS: Based on patient's epidemiological characteristics and bacteriological type, it was necessary to actively enact measures to control their recurrent.

Control of chronic obstructive pulmonary disease in urban populations: findings from a cross-sectional prevalence survey in Shenzhen, China.

The prevalence of chronic obstructive pulmonary disease (COPD) among urban populations is generally lower than rural residents, but the disease burden is still high. We conducted a cross-sectional prevalence survey of COPD among residents aged ≥40 years in an emerging city Shenzhen, China from September 2018 to June 2019. Through multi-stage stratified random sampling, a total of 4157 eligible participants were invited to complete a questionnaire and to take the spirometry test; 3591 with available data were enrolled in the final analysis. Individuals were diagnosed with COPD if the post-bronchodilator FEV1/FVC ratio was less than 0.7. The estimated standardized prevalence of COPD among residents over 40 years old in Shenzhen was 5.92% (95% confidential intervals [CI] 4.05-8.34). Risk factors for COPD included elder age (adjusted odds ratio 1.206, 95% CI 1.120-1.299 per 10-year increase), smoking over 20 pack-years (1.968, 1.367-2.832), history of chronic bronchitis (1.733, 1.036-2.900) or asthma (4.920, 2.425-9.982), and exposure to higher annual minimum concentrations of ambient SO2 (1.156, 1.053-1.270 per 1-µg/m3 increase). Among 280 spirometry-diagnosed patients, most (221, 78.93%) patients were classified as mild COPD (GOLD stage I). This survey found that the prevalence of COPD in Shenzhen is low and most patients had mild symptoms, thus recommended screening using spirometry in primary health care to detect early-stage COPD. Increased risk from the exposure to air pollutants also indicated the urgent need for environmental improvement in city settings.

Quantitative assessment of the effectiveness of joint measures led by Fangcang shelter hospitals in response to COVID-19 epidemic in Wuhan, China.

OBJECTIVE: To quantitatively evaluate the effectiveness of Fangcang shelter hospitals, designated hospitals, and the time interval from illness onset to diagnosis toward the prevention and control of the COVID-19 epidemic. METHODS: We used SEIAR and SEIA-CQFH warehouse models to simulate the two-period epidemic in Wuhan and calculate the time dependent basic reproduction numbers (BRNs) of symptomatic infected individuals, asymptomatic infected individuals, exposed individuals, and community-isolated infected individuals. Scenarios that varied in terms of the maximum numbers of open beds in Fangcang shelter hospitals and designated hospitals, and the time intervals from illness onset to hospitals visit and diagnosis were considered to quantitatively assess the optimal measures. RESULTS: The BRN decreased from 4.50 on Jan 22, 2020 to 0.18 on March 18, 2020. Without Fangcang shelter hospitals, the cumulative numbers of cases and deaths would increase by 18.58 and 51.73%, respectively. If the number of beds in the designated hospitals decreased by 1/2 and 1/4, the number of cumulative cases would increase by 178.04 and 92.1%, respectively. If the time interval from illness onset to hospital visit was 4 days, the number of cumulative cases and deaths would increase by 2.79 and 6.19%, respectively. If Fangcang shelter hospitals were not established, the number of beds in designated hospitals reduced 1/4, and the time interval from visiting hospitals to diagnosis became 4 days, the cumulative number of cases would increase by 268.97%. CONCLUSION: The declining BRNs indicate the high effectiveness of the joint measures. The joint measures led by Fangcang shelter hospitals are crucial and need to be rolled out globally, especially when medical resources are limited.

Changes in Incidence and Epidemiological Characteristics of Pulmonary Tuberculosis in Mainland China, 2005-2016.

Importance: The World Health Organization End TB (Tuberculosis) Strategy aims to decrease the global incidence and mortality of TB by 90% and 95%, respectively, as of 2035. Objective: To characterize the recent epidemiological trend of pulmonary TB (PTB) in mainland China based on the national surveillance data. Design, Setting, and Participants: This cross-sectional study collected demographic and clinical data of all patients reported in the national Tuberculosis Information Management System of China from January 1, 2005, to November 21, 2016. Data were analyzed from December 1, 2019, to July 31, 2020. Exposures: Pulmonary TB was defined as bacteriologically confirmed or clinically diagnosed TB in the lung parenchyma or the tracheobronchial tree. Main Outcomes and Measures: Temporal and spatial variation of annual incidence and demographic features of PTB in mainland China. Results: In total, 10 582 903 patients with PTB were reported in mainland China from 2005 to 2016. The median age of patients with PTB was 46 (interquartile range [IQR], 30-61) years, and 28.53% were 60 years or older. Most patients with PTB were male (69.8%) and farmers or herders (70.0%). The mean (SD) incidence of PTB was 66.61 (8.09) per 100 000 population. The annual incidence decreased from 72.95 per 100 000 population in 2005 to 52.18 per 100 000 population in 2016, and the reduction was greater in the eastern and central regions (31.6%; from 69.43 to 47.48 per 100 000 population) than in the western region (21.0%; from 82.06 to 64.82 per 100 000 population). Xinjiang Uygur Autonomous Region (135.03 per 100 000 population), Guizhou Province (115.98 per 100 000 population), and the Tibet Autonomous Region (101.98 per 100 000 population) had the highest mean annual incidences. The median time from onset of illness to diagnosis decreased from 36 (IQR, 16-92) days from 2005 to 2007 to 31 (IQR, 15-63) days in 2008 and later (P < .001) and was longer in the western region than in the eastern and central regions (41 [IQR, 20-91] vs 30 [IQR, 13-61] days; P < .001). Conclusions and Relevance: Although this study found that the incidence of PTB in mainland China showed a downward trend from 2005 to 2016, to achieve the World Health Organization 2035 goal, innovative and more efficient prevention and control strategies are needed, particularly among the most susceptible population, that is, farmers and herders in western China.

Assessment of Strategies and Epidemiological Characteristics of Tuberculosis in Henan Province, China: Observational Study.

BACKGROUND: In 2005, China established an internet-based Tuberculosis Information Management System (TBIMS) to monitor changes in tuberculosis (TB). Many scholars have conducted epidemiological research using TBIMS; however, few studies assessing control strategies have been performed based on this platform data. Henan province is a high TB incidence area in China where, in addition to following the nationwide TB strategies, a series of local intervention combinations have been implemented. OBJECTIVE: Our study aims to evaluate the impact of nationwide TB intervention combinations on epidemiological changes and determine whether Henan province can achieve the World Health Organization's (WHO) goal of reducing TB incidence by 50% and TB mortality by 75% by the year 2025. METHODS: We used descriptive statistical methods to show the spatial and temporal distribution of pulmonary tuberculosis (PTB) reported to the TBIMS database from 2005 to 2018, and logistic regression analysis was performed to identify the risk factors of bacteriological-positive TB. The dynamic compartmental model and Bayesian melding approach was adopted to estimate the burden of TB under the impact of different TB control policies. RESULTS: In total, 976,526 PTB cases were notified to the TBIMS in Henan in a period of 14 years. Although the overall incidence of PTB declined from 91.4/105 to 58.5/105, and the overall incidence of bacteriological-positive PTB declined from 44.5/105 to 14.7/105, the WHO's 2025 goal could not be met. The distribution of high incidence and poverty-stricken counties were basically overlapped. Men, farmers and herdsmen (in rural areas), and subjects aged ≥60 years were more likely to develop bacteriological-positive PTB. The increasing treatment success for drug-susceptible tuberculosis and multidrug-resistant tuberculosis has not provided the desired reduction in incidence and mortality. CONCLUSIONS: To achieve the targeted goal, while improving the cure rate of TB, new active (rather than passive) detection and intervention strategies should be formulated based on epidemiological characteristics in Henan province.

Associated factors of co-existent pulmonary tuberculosis and lung cancer: A case-control study.

BACKGROUND: Although the incidence of co-existent pulmonary tuberculosis (PTB) and lung cancer in China is increasing, risk factors related to its development are still poorly understood. We aimed to investigate which clinical factors were associated with the odds of co-existent PTB and lung cancer (PTB-lung cancer) cases in a case-control study. METHOD: A total of 125 PTB-lung cancer patients were enrolled by Beijing Chest Hospital as the case group between January 2012 and December 2016. Age- and sex-matched PTB-only (N = 125) and lung cancer-only (N = 125) patients were selected as the control groups. Data were collected from the medical records and computed tomography (CT) reports. The case group was further categorized into three sub-groups according to the diagnosis intervals between previous PTB and lung cancer (<1 year, 1-10 years, and > 10 years). RESULT: Compared with both controls of PTB-only and lung cancer-only patients, the PTB-lung cancer case group had significantly higher proportions of patients with irritant cough, expectoration, hemoptysis, fever and CT features of irregular mass and pleural thickening. For PTB patients, fibrous calcification (OR, 2.193; 95%CI, 1.168-4.117) was associated with higher odds of lung cancer (P-value < .05). CONCLUSION: Distinct clinical symptoms and CT tests may help with the early diagnosis of PTB-lung cancer cases. PTB patients with fibrous calcification may have a higher risk of lung cancer. Further multicenter prospective studies are required to validate our findings.

The epidemiology of pulmonary tuberculosis in children in Mainland China, 2009-2015.

OBJECTIVE: To describe the incidence and epidemiological characteristics of child pulmonary tuberculosis (PTB) notified to the Chinese Center for Disease Control and Prevention (CDC) Tuberculosis Information Management System (TBIMS) from 2009 to 2015. STUDY DESIGN: A database of child PTB for 31 provinces in mainland China over 7 years was retrospectively analysed. The spatiotemporal distribution of child PTB was described. RESULTS: In total, 35,710 child PTB cases were notified to the TBIMS, representing only 0.60% of all 5,991,593 PTB case notifications. The average annual notified incidence of child PTB was 2.44/ per 100,000 children (95% CI: 1.77-3.10) and decreased by 52.53% in all age groups during the study period. Tibet had the highest incidence (15.95/ per 100,000 children), followed by Guizhou and Xinjiang. However, the case numbers were the most in Guizhou. The 0-1 year and 12-14 years of age groups exhibited the most cases. The positive rates of sputum smears, bacteriology and chest X-rays abnormality were 21.5%, 21.7% and 98.1%, respectively, which were lower than those 15 years of age and older (all p-values<0.0001). CONCLUSION: The notified incidence of child PTB in mainland China decreased substantially over 7 years. Future prevention and control of PTB in children should focus on the 0-1 and 12-14 years of age groups, and Tibet and Guizhou provinces. However, the notified incidence is still low, relative to adults, suggesting substantial under-reporting. Thus, more effective care seeking, identification and registration of children with TB are crucial.

Screening candidate microRNAs (miRNAs) in different lambskin hair follicles in Hu sheep.

Hu sheep lambskin is a unique white lambskin from China that exhibits three types of flower patterns, including small waves, medium waves, and large waves, with small waves considered the best quality. However, our understanding of the molecular mechanism underlying flower pattern formation in Hu sheep lambskin is limited. The aim of the present study was to further explore the relevance between candidate microRNAs (miRNAs) and developmental characteristics of hair follicles and screen miRNAs for later functional validation. Herein, we employed Illumina Hiseq 2500 to identify differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves to construct a comprehensive sequence database on the mechanism of hair follicle development. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of candidate miRNAs in different flower patterns were analyzed by relative quantitation using real-time PCR, combined with histological observation and micro-observation technologies, and the correlation between expression levels of candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0. A total of 522 differentially expressed miRNAs were identified, and RNA-seq analysis detected 7,266 target genes in different groups of flower patterns. Gene ontological analysis indicated these target genes were mainly involved in cell proliferation, differentiation, growth, apoptosis, and ion transport, and 14 miRNAs, including miR-143, miR-10a, and let-7 were screened as candidate miRNAs in Hu sheep hair follicle growth and development. In the same field of vision, variance analysis showed that the number of secondary follicles in small waves was significantly larger than that in large and medium waves (P<0.01); the diameter of the primary and secondary follicles in large waves was respectively larger than those in medium and small waves (P<0.01). Combined with correlation analysis between miRNA expression and histological properties of hair follicles, highly significant differences in miRNA-143 expression levels between large and small waves were observed (P<0.01), and significant differences in the miRNA-10a expression levels between large and small waves (P<0.05) and in let-7i expression levels between large and medium waves were observed (P<0.05). Significant differences in the expression of novel miRNAs of NW_004080184.1_6326 between medium and large waves were detected (P<0.05), and highly significant differences between medium and small waves were observed (P<0.01). Highly significant differences in the expression level of NW_004080165.1_8572 between medium and large and small waves (P<0.01), in that of NW_004080181.1_3961 between medium and small waves (P<0.01), and in that of NW_004080190.1_13733 between medium and large waves were observed, whereas no significant differences in the other miRNAs among large, medium, and small waves were detected. Overall, the present study showed that miRNA-143, miRNA-10a, let-7i, NW_004080184.1_6326, NW_004080165.1_8572, NW_004080181.1_3961, and NW_004080190.1_13733 could be considered as important candidate genes, indicating these seven miRNAs may play significant roles in hair follicle growth and development in Hu sheep lambskin.

An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep.

Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep's wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium waves included 13 downregulated miRNAs that had regulatory effects on 64 upregulated genes and 4 upregulated miRNAs, which in turn had regulatory effects on 22 downregulated genes. Compared to medium waves, large waves consisted of 13 upregulated miRNAs that had regulatory effects on 48 downregulated genes. These differentially expressed miRNAs and genes may play a significant role in forming different patterns, and provide evidence for the molecular mechanisms underlying the formation of hair follicles of varying patterns.