Functional categorization of gene regulatory variants that cause Mendelian conditions.

Much of our current understanding of rare human diseases is driven by coding genetic variants. However, non-coding genetic variants play a pivotal role in numerous rare human diseases, resulting in diverse functional impacts ranging from altered gene regulation, splicing, and/or transcript stability. With the increasing use of genome sequencing in clinical practice, it is paramount to have a clear framework for understanding how non-coding genetic variants cause disease. To this end, we have synthesized the literature on hundreds of non-coding genetic variants that cause rare Mendelian conditions via the disruption of gene regulatory patterns and propose a functional classification system. Specifically, we have adapted the functional classification framework used for coding variants (i.e., loss-of-function, gain-of-function, and dominant-negative) to account for features unique to non-coding gene regulatory variants. We identify that non-coding gene regulatory variants can be split into three distinct categories by functional impact: (1) non-modular loss-of-expression (LOE) variants; (2) modular loss-of-expression (mLOE) variants; and (3) gain-of-ectopic-expression (GOE) variants. Whereas LOE variants have a direct corollary with coding loss-of-function variants, mLOE and GOE variants represent disease mechanisms that are largely unique to non-coding variants. These functional classifications aim to provide a unified terminology for categorizing the functional impact of non-coding variants that disrupt gene regulatory patterns in Mendelian conditions.

[Laparoscopic surgery in treatment of perivascular epithelioid cell tumor in liver: safety and efficacy].

Objective: To evaluate the efficacy and safety of laparoscopic surgery for perivascular epithelioid cell tumor (PEComa). Methods: The clinical data of 42 patients with hepatic PEComa diagnosed by pathology in Hunan Provincial People's Hospital from September 2012 to September 2021 were retrospectively analyzed. The patients were divided into the endoscopic group and the open group according to surgical methods. Statistical software was used to compare the differences in operation time, intraoperative blood loss, postoperative hospital stay, postoperative pathological data and incidence of complications between the two groups. Results: There were 27 cases in the endoscopic group and 15 cases in the open group. In the endoscopic group, there were 5 males and 22 females, aged (40.0±10.4) years. In the open group, there were 5 males and 10 females, aged (44.5±12.6) years. The operative time of the endoscopic group and the open group was (239±156.2) min and (348±103.0) min, and the postoperative hospital stay was (8.2±2.4) d and (13.7±4.9) d, respectively, the endoscopic group was significantly better than the open group, and the difference was statistically significant (P<0.05). There was no significant difference in intraoperative blood loss, tumor benign and malignant, tumor site, tumor diameter, Ki67 index, postoperative complications such as biliary leakage, incision infection and pleural effusion (P>0.05). During the follow-up period of 2-103 months, one case was lost to follow-up, two cases died in the endoscopic group,one case died in the open group. The 5-year overall survival rate (OS) and disease-free survival rate (DFS) were 80.8% and 83.0%, respectively. Meanwhile,. The 5-year OS and DFS were both 92.3%, the difference was not statistically significant (P>0.05). Conclusions: Laparoscopic treatment of hepatic PEComa has the advantages of short operation time and short postoperative hospital stay, and the incidence of complications, 5-year OS and DFS are not significantly different from that of the open group.

[Analysis of the diagnosis and treatment of 24 cases of hepatic perivascular epithelioid cell tumor].

Objective: To investigate hepatic perivascular epithelioid cell tumor (PEComa) diagnosis and treatment plan. Methods: 24 cases diagnosed with PEComa clinical manifestations, serum alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), imaging findings, surgical methods, postoperative hospital stay, pathological results and prognosis were analyzed retrospectively from September 2015 to September 2020. Results: Majority of patients were females (79.2%), aged 41.5±11.4 years. Tumors were predominantly located in the right liver (50.0%). 76.7% of the cases were mostly clinically asymptomatic. AFP, CEA and CA199 indices were all negative. CT mostly showed low density tumor in the plain scan phase, enhanced in the enhancement phase, and enhanced and weakened in portal venous and equilibrium phase (66.7%). MRI manifestations of most tumors were hypointense on T1WI and hyperintense on T2WI (72.7%). B-ultrasound mostly showed hyperechoic mass in the tumor area with punctate vascular shadow (52.9%). Postoperative hospital stay was 9.0±2.4 days for laparoscopic surgery patients (n=13), 13.4±6.3 days for traditional laparotomy (hereinafter referred to as laparotomy, n=10), and 3 days for 1 patient with microwave ablation. All postoperative pathological results were positive for HMB45 and Melan-A. Follow-up results: 21 cases survived normally, with no tumor recurrence in the recent physical examination; two cases had tumor recurrence and they died two and three years after surgery, and one case was lost to follow-up. Conclusion: Hepatic PEComa more commonly occurs in middle-aged women, with no specific features for tumor markers and clinical manifestations. Some imaging findings are specific, so its features can be combined as a basis for diagnosis. Postoperative pathological examination results can confirm the diagnosis. Therefore, surgery remains the initial treatment plan. Microwave ablation and laparoscopic surgery are recommended as the preferred option because of shorter hospital stays and less trauma than open surgery.

[Mucinous adenocarcinoma of the gallbladder: report of 5 cases].

The clinical manifestations, laboratory and imaging results, diagnosis and treatment and prognosis of 5 patients with mucinous adenocarcinoma of the gallbladder were analyzed. It was found that the clinical characteristics of mucinous adenocarcinoma of the gallbladder were very similar to that of gallbladder malignant tumor, and it was difficult to differentiate. Ultrasound guided gallbladder needle biopsy can confirm the diagnosis before surgery. Surgery is still the first choice of treatment. Since laparoscopic surgery has obvious advantages over open surgery, it is recommended to be preferentially used. If the tumor is under T2 stage and has no aggressive metastasis, the prognosis is generally good.

[Cross-reaction of seasonal influenza vaccine immune serum against Eurasian Avian-like H1N1 swine influenza virus].

Objective: To evaluate the cross-reaction of seasonal influenza vaccine immune serum against Eurasian avian-like H1N1 swine influenza virus. Methods: Nine human infected Eurasian avian-like H1N1 swine influenza virus strains were obtained from national influenza surveillance network laboratories in Jiangsu, Hebei, Shandong, Yunnan, Hunan, Fujian and Tianjin provinces, and their genetic characteristics of hemagglutinin were analyzed by deep sequencing. 30 volunteers were recruited respectively from children (2-5 years old), adults (24-57 years old) and elderly (60-84 years old) who received 2019-2020 seasonal influenza vaccine in Anning city, Yunnan Province in October 2019, and serum samples were collected before and 1 month after vaccination. The hemagglutination inhibition test was used to evaluate the cross-reaction of serum before and after immunization against 4 strains of human infection with Eurasian avian-like H1N1 swine influenza virus isolated since 2015. Results: The homology of hemagglutinin genes of 9 Eurasian avian-like H1N1 swine influenza viruses was similar, but the difference of hemagglutinin heavy chain and light chain amino acid genes with A (H1N1) pdm09 (vaccine strain) were 90-101 and 24-30 amino acids respectively. The antibody titer of vaccine strain antiserum to vaccine strain was 2 560; the antibody titers of the vaccine strain antiserum to Eurasian avian-like H1N1 swine influenza virus and the Eurasian avian-like H1N1 swine influenza virus antiserum to vaccine strain were same as 640. The proportion of children, adults and elderly vaccinated with seasonal influenza vaccine with antibody titer ≥40 against vaccine strain was 90.0%, 70.0% and 73.3%, respectively; while the proportion merely were 46.7%, 36.7% and 33.3%-43.3% to 4 strains of Eurasian avian-like H1N1 swine influenza virus, respectively. Conclusion: Seasonal influenza vaccination does not provide effective cross-protection against Eurasian avian-like H1N1 swine influenza virus.

[The malignant phenotype of calcineurin B homologous protein 2 in gastric cancer and its clinical significance].

Objective: To study the expression and clinical significance of calcineurin B homologous protein 2 (CHP2) in gastric cancer (GC) and its effect on malignant phenotype of GC cells. Methods: The protein expression of CHP2 in 297 GC tissue and 198 normal gastric tissue samples were detected by immunohistochemistry. The relationship between the expression of CHP2 and clinicopathologic parameters of GC were analyzed. CHP2-overexpression plasmids and CHP2-interference plasmids were transfected into GC cell lines respectively. Wound healing assay and Transwell experiment was used to detect the invasion and migration ability of GC cells, and cell counting kit-8 (CCK-8) method was used to detect the proliferation ability of GC cells. Results: The positive expression rate of CHP2 in GC was 68.7% (204/297), which was higher in benign margin (34.1%) (31/91), chronic gastritis (59.1%) (13/22), intestinal metaplasia (34.2%) (13/38), low-grade intraepithelial neoplasia (40.0%) (12/30) and high-grade intraepithelial neoplasia (41.2%)(7/17). The positive expression of CHP2 was correlated with tumor, node and metastasis (TNM) stage, lymph node metastasis and distant metastasis (all P<0.05), but not with gender, age, Laurén classification, human epidermal growth factor receptor 2 (HER2) levels, depth of invasion, carcinoembryonic antigen (CEA) level and CEA 19-9 level (all P>0.05). The results of multivariate analysis showed that high expression of CHP2 and TNM stage were both independent parameters for predicting GC patient prognosis (both P<0.05). Interference of CHP2 expression in HGC-27 cells suppressed proliferation and migration significantly (P<0.05). However, over-expression CHP2 in AGS cells promoted proliferation, and migration significantly (P<0.05). Conclusion: CHP2 plays an important role in the development of GC, which is expected to be a molecular marker for patient prognosis and a potential target of targeted therapy for GC patients.

[The clinical significance of Th17 cells in patients with myelodysplastic syndrome and iron overload].

Objective: To study the significance of Th17 cells in patients with myelodysplastic syndrome (MDS) and iron overload. Methods: A total of 77 patients with MDS admitted to Guangzhou First People's Hospital were enrolled from January 2017 to December 2018,who were divided into iron overload group (37 cases) with serum ferritin (SF) over 1000 µg/L and non-ferrous overload group(40 cases). CD(4)(+)T cells in peripheral blood (PB) and bone marrow (BM) were sorted by flow cytometry. The ratio of Th17 cells and cells with abnormal karyotype were compared. IL-17 and IL-6 protein and RNA expression were detected by ELISA and quantitative real-time PCR(qRT-PCR). Results: The proportions of Th17 cells in PB and BM in iron overload group were significantly higher than those in non-iron overload group [(41.06±0.96)% vs. (26.80±1.21)%; (47.39±1.60)% vs. (34.29±1.03)%; P<0.01]. The Th17 positive cells with abnormal karyotype in iron overload group were more than those in non-iron overload group[(4.96±0.53)% vs. (3.67±0.12)% in PB; (10.06±1.67)% vs. (4.36±0.43)% in BM; P<0.01]. Similarly,the protein levels as well as mRNA expression of IL-6 and IL-17 in patients with iron overload were significantly higher than those in non-iron overload group (P<0.01 both in PB and BM). Conclusions: As hematopoietic regulators secreted by Th17 cells, the expression of IL-6 and IL-17 in MDS patients with iron overload are elevated. This may predict the influence of these factors to the differentiation of Th17 cells.

Characterization and virus susceptibility of a continuous cell line derived from the brain of Aequidens rivulatus (Günther).

Cell cultures derived from the brain tissues of Aequidens rivulatus (Günther) have been characterized previously. In this study, a continuous cell line ARB8 was further established, and its growth characteristics, transcription and susceptibility to fish viruses-including chum salmon reovirus (CSV), marbled eel infectious pancreative necrosis virus (MEIPNV), grouper nervous necrosis virus (GNNV), giant seaperch iridovirus (GSIV), red seabream iridovirus (RSIV), koi herpesvirus (KHV), herpesvirus anguilla (HVA) and marbled eel polyoma-like virus (MEPyV)-were examined. ARB8 cells that showed epithelioid morphology and were passaged >80 times grew well at temperatures ranging from 25°C to 30°C in L-15 medium containing 5%-15% foetal bovine serum. The cells constitutively transcribed connexion 43, glutamine synthetase, nestin and nkx6-2, which are markers for neural progenitor cells. The cells were highly susceptible to CSV, MEIPNV, GSIV and RSIV and showed the typical cytopathic effect (CPE). However, the cells were resistant to GNNV, KHV, HVA and MEPyV because no significant CPE was noted after infection. Optimal temperatures for virus production ranged from 25°C to 30°C. The results revealed that the neural progenitor cell line ARB8 can potentially serve as a useful tool for investigating fish viruses and isolating new viruses in ornamental cichlid fishes.

An impact of Deoxynivalenol produced by Fusarium graminearum on broiler chickens.

Deoxynivalenol (DON) is a major mycotoxin from the trichothecene family of mycotoxins produced by Fusarium fungi that cause disease in grains. Food and feed contaminated with DON cause a variety of adverse health effects in humans and farm animals. However, the fermentation conditions of DON for toxicological study remain to be optimised. Deoxynivalenol levels were increased in a constant temperature of 20°C after fermentation for 7 and 14 days compared with cycling temperatures between 10-20°C and 15-20°C. We have established that optimum conditions for DON production on rice-based medium is constant temperature at 20°C. In feeding experiments, the villus height and the ratio of villus height and crypt depth were reduced in the duodenum of DON-fed broiler chickens, whereas a mycotoxin degradation agent efficiently reversed the abnormal morphology of the small intestine in the DON-fed broilers. The expression of pro-inflammatory gene, COX-2, was induced in the spleen and bursa of Fabricius of DON-fed broilers. This effect was alleviated in the presence of mycotoxin degradation agent. DON produced in solid-state fermentation is able to cause toxic effects in broilers and induce an abnormal morphology of the small intestine, particularly the duodenum.

[Exposure to disinfection by-products during gestation and newborns' small for gestational age].

Objective: To explore the associations between exposure to chlorination disinfection by-products (CDBPs) during gestation and newborns' small for gestational age (SGA). Methods: During April 2010 to July 2012, a total of 3 903 pregnant women who lived in a district with the same water treatment plant in Wuhan, China were recruited to this perspective study. Information about demographic characteristics of pregnant women and their newborns was collected. The tap water samples were monthly collected for 28 months in 3 different sites, with 84 samples, and 4 kinds of trihalomethanes (THMs)(chloroform (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and bromoform (TBM)) and 2 kinds of chlorohaloacetic acids (HAAs) (trichloroacetic acid (TCAA), dichloroacetic acid (DCAA)) were determined. The pregnant women were divided into 4 groups(Q1 to Q4) by quartile method according to their exposure level of CDBPs. Binary Logistic regression models were used to assess the associations between exposure to CDBPs during gestation and newborns' small for gestational age. Results: The average weight of all the newborns was (3 310.19±389.91) g, of which 169 (4.33%) were SGA. The median concentrations of TCM, BDCM, bromo-THMs, total THMs, TCAA, and DCAA during the whole pregnancy were 18.07, 4.93, 8.51, 26.74, 10.65, and 13.77 µg/L, respectively. Binary Logistic regression analysis showed dose-response relationships between elevated TCM and total THMs during the whole gestation and compared with Q1 group, while there was a increased risk of SGA in Q4 group, and OR(95%CI) was 1.87 (1.01-3.49) , 2.30 (1.22-4.35) , respectively (P for trend equaled to 0.044, 0.015). Compare with Q1 group, there also be positive associations between exposure to TCAA (Q4 group) during first-trimester and the whole gestation and SGA, while OR(95%CI) was 2.16 (1.19-3.91) (P for trend equaled to 0.015). Conclusion: Exposure to CDBPs during gestation might increase the risk of newborns' SGA.

[Expression of succinate dehydrogenase subunit protein in succinate dehydrogenase-deficient gastrointestinal stromal tumors].

Objective: To investigate the expression of succinate dehydrogenase complex subunit protein in succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs). Methods: Three hundred fifty-two cases of GISTs were collected from January 2003 to January 2017 at the Affiliated Hospital of Guizhou Medical University and West China Hospital of Sichuan University.The expression of succinate dehydrogenase subunit protein was detected by immunohistochemical EnVision technique in 352 cases of GISTs, and the negative cases were analyzed for clinicopathologic features and outcome. The gene segments of CKIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were amplified and detected in SDH-deficient (negative) cases. Results: A total of 15 SDHB-deficient (negative) GISTs (4.3%, 15/352) were found among 352 cases of GISTs. Six patients were male and nine were female. The age of initial diagnosis ranged from 15 to 84 years (median=53 years, mean=47 years). The tumor involved stomach (14 cases) and mesentery (1 case). The tumor sizes varied from 0.5 cm to 15.0 cm (mean=6.9 cm). There were six, six and three cases of epithelioid, mixed and spindle cell types respectively. Eight cases showed multi-nodularity in the wall of stomach. Metastasis to lymph node was noted in four cases, one case showed intraperitoneal implantation metastasis. Metastases to liver, pancreas and lymph node were found in one case, and one case showed vascular invasion. Among SDHB-deficient GISTs, two SDHA-deficient (negative) cases were found (0.6%, 2/352), but there were no SDHC and SDHD deficient (negative) cases. Five of the fifteen SDH-deficient GISTs had follow-up data: one patient died 8 months after surgery from unknown cause, four had no recurrences or metastases, and there was no history of paraganglioma and pulmonary chondroma found in patients and their families. No mutation in CKIT and PDGFRA gene was identified in 15 cases of SDH-deficient GISTs. Conclusion: SDH-deficient GISTs have unique clinicopathologic features and a favorable prognosis, and a small proportion of cases are SDHA-deficient.

Assessing coughing-induced influenza droplet transmission and implications for infection risk control.

Indoor transmission of respiratory droplets bearing influenza within humans poses high risks to respiratory function deterioration and death. Therefore, we aimed to develop a framework for quantifying the influenza infection risk based on the relationships between inhaled/exhaled respiratory droplets and airborne transmission dynamics in a ventilated airspace. An experiment was conducted to measure the size distribution of influenza-containing droplets produced by coughing for a better understanding of potential influenza spread. Here we integrated influenza population transmission dynamics, a human respiratory tract model, and a control measure approach to examine the indoor environment-virus-host interactions. A probabilistic risk model was implemented to assess size-specific infection risk for potentially transmissible influenza droplets indoors. Our results found that there was a 50% probability of the basic reproduction number (R0) exceeding 1 for small-size influenza droplets of 0·3-0·4 µm, implicating a potentially high indoor infection risk to humans. However, a combination of public health interventions with enhanced ventilation could substantially contain indoor influenza infection. Moreover, the present dynamic simulation and control measure assessment provide insights into why indoor transmissible influenza droplet-induced infection is occurring not only in upper lung regions but also in the lower respiratory tract, not normally considered at infection risk.

Evaluation of Relative Bioavailability of 25-Hydroxycholecalciferol to Cholecalciferol for Broiler Chickens.

This study was conducted to evaluate the relative bioavailability (RBV) of 25-hydroxycholecalciferol (25-OH-D3) to cholecalciferol (vitamin D3) in 1- to 21-d-old broiler chickens fed with calcium (Ca)- and phosphorus (P)-deficient diets. On the day of hatch, 450 female Ross 308 broiler chickens were assigned to nine treatments, with five replicates of ten birds each. The basal diet contained 0.50% Ca and 0.25% non-phytate phosphorus (NPP) and was not supplemented with vitamin D. Vitamin D3 was fed at 0, 2.5, 5.0, 10.0, and 20.0 µg/kg, and 25-OH-D3 was fed at 1.25, 2.5, 5.0, and 10.0 µg/kg. The RBV of 25-OH-D3 was determined using vitamin D3 as the standard source by the slope ratio method. Vitamin D3 and 25-OH-D3 intake was used as the independent variable for regression analysis. The linear relationships between the level of vitamin D3 or 25-OH-D3 and body weight gain (BWG) and the weight, length, ash weight, and the percentage of ash, Ca, and P in femur, tibia, and metatarsus of broiler chickens were observed. Using BWG as the criterion, the RBV value of 25-OH-D3 to vitamin D3 was 1.85. Using the mineralization of the femur, tibia, and metatarsus as criteria, the RBV of 25-OH-D3 to vitamin D3 ranged from 1.82 to 2.45, 1.86 to 2.52, and 1.65 to 2.05, respectively. These data indicate that 25-OH-D3 is approximately 2.03 times as active as vitamin D3 in promoting growth performance and bone mineralization in broiler chicken diets.

Sharp resonance with complete transmission in Thue-Morse microring resonators.

We present sharp resonances with complete transmission in the Thue-Morse microring resonator. The quasi-periodic structure attains a better quality of filtering than the traditional periodic structure. This includes the following: (i) the high-Q resonances with complete transmission are obtained in the transmission spectra, (ii) the maximum value of transmittance always remains 1 for an arbitrary proportion of two different radiuses in the major-band regions, and (iii) the complete transmission occurs for arbitrary coupling in the mini-band regions. Moreover, we propose an analytical method to predict the occurrences of resonance peaks in the mini-band regions based on the gap map.

Extraordinary light absorptance in graphene superlattices.

Extraordinary absorption decrease in graphene superlattices in the visible range is presented. Due to competition between loss and resonant reflection at resonance, the absorption displays non-monotonic behavior. As the period number increases above a certain critical value, absorption decreases with the increase in the period number. This is in contrast to ordinary absorption for a non-resonant condition, which monotonically increases with the period number. Moreover, this extraordinary property can also be controlled by applying a gate voltage to graphene sheets. The results provide not only a new understanding of graphene physics but also an application in nanophotonics and optoelectronics.

Statistically background-free, phase-preserving parametric up-conversion with faint light.

We demonstrate up-conversion with no statistically significant background photons and a dynamic range of 15 decades. Near-infrared 920 nm photons were converted into the visible at 577 nm using periodically poled lithium niobate waveguides pumped by a 1550 nm laser. In addition to achieving statistically noiseless frequency up-conversion, we report a high degree of phase preservation (with fringe visibilities ≥ 0.97) at the single-photon level using an up-converting Mach-Zehnder interferometer. This background-free process opens a path to single-photon detection with no intrinsic dark count. Combined with a demonstrated photon-number preserving property of an up-converter, this work demonstrates the feasibility of noiseless frequency up-conversion of entangled photon pairs.

High-order micro-ring resonator with perfect transmission using symmetrical Fibonacci structures.

A symmetrical Fibonacci micro-ring resonator (SFMR) has been presented to avoid the coupled resonator optical waveguide (CROW) bottle, which is a bottle-shaped distribution for high orders in transmission spectra. The SFMR features three advantages that improve filtering quality compared to that provided by traditional periodic micro-ring resonators. First, sharper resonances are obtained by eliminating the CROW bottle from the mini gaps that appear in the major-band region. Second, peaks with perfect transmission are always obtained without a radius and coupling modulation in the mini-band regions and major-band regions. Third, the full width at half-maximum of the band-edge peak decreases with the increasing generation order.

Network information analysis reveals risk perception transmission in a behaviour-influenza dynamics system.

Influenza poses a significant public health burden worldwide. Understanding how and to what extent people would change their behaviour in response to influenza outbreaks is critical for formulating public health policies. We incorporated the information-theoretic framework into a behaviour-influenza (BI) transmission dynamics system in order to understand the effects of individual behavioural change on influenza epidemics. We showed that information transmission of risk perception played a crucial role in the spread of health-seeking behaviour throughout influenza epidemics. Here a network BI model provides a new approach for understanding the risk perception spread and human behavioural change during disease outbreaks. Our study allows simultaneous consideration of epidemiological, psychological, and social factors as predictors of individual perception rates in behaviour-disease transmission systems. We suggest that a monitoring system with precise information on risk perception should be constructed to effectively promote health behaviours in preparation for emerging disease outbreaks.

Assessing dengue infection risk in the southern region of Taiwan: implications for control.

Dengue, one of the most important mosquito-borne diseases, is a major international public health concern. This study aimed to assess potential dengue infection risk from Aedes aegypti in Kaohsiung and the implications for vector control. Here we investigated the impact of dengue transmission on human infection risk using a well-established dengue-mosquito-human transmission dynamics model. A basic reproduction number (R 0)-based probabilistic risk model was also developed to estimate dengue infection risk. Our findings confirm that the effect of biting rate plays a crucial role in shaping R 0 estimates. We demonstrated that there was 50% risk probability for increased dengue incidence rates exceeding 0.5-0.8 wk-1 for temperatures ranging from 26°C to 32°C. We further demonstrated that the weekly increased dengue incidence rate can be decreased to zero if vector control efficiencies reach 30-80% at temperatures of 19-32°C. We conclude that our analysis on dengue infection risk and control implications in Kaohsiung provide crucial information for policy-making on disease control.