Development of a nine-variant reference material panel to standardize cell-free DNA detection.

Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.

Reliable biological and multi-omics research through biometrology.

Metrology is the science of measurement and its applications, whereas biometrology is the science of biological measurement and its applications. Biometrology aims to achieve accuracy and consistency of biological measurements by focusing on the development of metrological traceability, biological reference measurement procedures, and reference materials. Irreproducibility of biological and multi-omics research results from different laboratories, platforms, and analysis methods is hampering the translation of research into clinical uses and can often be attributed to the lack of biologists' attention to the general principles of metrology. In this paper, the progresses of biometrology including metrology on nucleic acid, protein, and cell measurements and its impacts on the improvement of reliability and comparability in biological research are reviewed. Challenges in obtaining more reliable biological and multi-omics measurements due to the lack of primary reference measurement procedures and new standards for biological reference materials faced by biometrology are discussed. In the future, in addition to establishing reliable reference measurement procedures, developing reference materials from single or multiple parameters to multi-omics scale should be emphasized. Thinking in way of biometrology is warranted for facilitating the translation of high-throughput omics research into clinical practices.

Effect of respiratory training on swallowing function in swallowing disorders: a systematic review and meta-analysis.

PURPOSE: To determine the clinical efficacy of different respiratory training interventions on swallowing function in patients with swallowing disorders through the systematic review. METHODS: We reviewed the literature regarding the application of respiratory training therapy in patients with swallowing disorders, followed by a PRISMA search of published literature in five databases (PubMed, Web of Science, The Cochrane Library, CINAHL and EMBASE) in December 2022. Two reviewers performed study selection, quality evaluation, and risk of bias, followed by data extraction and detailed analysis. RESULTS: A total of six randomized controlled studies with a total sample size of 193 cases were included. Respiratory training improved swallowing safety (PAS (n = 151, SMD = 0.69, 95% CI - 1.11 to - 0.26, I2 = 36, p < 0.001)) and swallowing efficiency [residual (n = 63, SMD = 1.67, 95% CI - 2.26 to - 1.09, I2 = 23%, p < 0.001)] compared to control groups. The results of the qualitative analysis conducted in this study revealed that respiratory training enhanced hyoid bone movement but had no effect on swallowing quality of life. CONCLUSIONS: Respiratory training interventions may improve swallowing safety and efficiency in patients with dysphagia. However, the level of evidence is low, and there is a limited amount of research on the effectiveness and physiology of this intervention to improve swallowing function. In the future, there is a need to expand clinical studies, standardize measurement tools, and improve study protocols.

Reference material development for detection of human respiratory syncytial virus using digital PCR.

Nucleic acid testing is a powerful tool for the detection of various pathogens. Respiratory syncytial virus (RSV) is a major cause of acute respiratory infection, especially in young children and infants. To improve the confidence and reliability of nucleic acid testing results for RSV, reference materials (RMs) of both type A and B of RSV were developed by the National Institute of Metrology, China, code numbers NIM-RM 4057 and 4058. The reference material was composed of in vitro transcribed RNA containing the nucleocapsid (N) gene, matrix (M) gene, and partial polymerase (L) gene of RSV. A duplex reverse transcription digital PCR method was established with limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) of 2, 5, and 23 copies per reaction for RSV-A and 4, 8, and 20 copies per reaction for RSV-B. The certified value and expanded uncertainty (U, k = 2) of the two RMs were determined to be (6.1 ± 1.4) × 104 copies/µL for RSV-A and (5.3 ± 1.2) × 104 copies/µL for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays.

Development of highly accurate digital PCR method and reference material for monkeypox virus detection.

Human monkeypox has attracted attention recently. Monkeypox virus (MPXV) keeps evolving as it spreading around the world rapidly, which may threaten the health of more and more people. Here, we have developed a high order reference method based on digital PCR (dPCR) for MPXV detection, of which the limits of quantification (LoQ) and detection (LoD) are 38 and 6 copies/reaction, respectively. Pseudovirus reference materials (RM) containing the conserved F3L gene has been developed, and the homogeneity assessment showed that the RM was homogeneous. The reference value with its expanded uncertainty determined by the established dPCR is (2.74 ± 0.46) × 103 copies/µL. Six different MPXV test kits were accessed by the RM. Four out of six test kits cannot reach their claimed LoDs. The poor analytical sensitivity might cause false-negative results, which lead to incorrect diagnosis and treatment. The establishment of a high order reference method of dPCR and pseudovirus RM is very useful for improving the accuracy and reliability of MPXV detection.

A culture-free method for rapidly and accurately quantifying active SARS-CoV-2.

Determining the quantity of active virus is the most important basis to judge the risk of virus infection, which usually relies on the virus median tissue culture infectious dose (TCID50) assay performed in a biosafety level 3 laboratory within 5-7 days. We have developed a culture-free method for rapid and accurate quantification of active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by targeting subgenomic RNA (sgRNA) based on reverse transcription digital PCR (RT-dPCR). The dynamic range of quantitative assays for sgRNA-N and sgRNA-E by RT-dPCR was investigated, and the result showed that the limits of detection (LoD) and quantification (LoQ) were 2 copies/reaction and 10 copies/reaction, respectively. The delta strain (NMDC60042793) of SARS-CoV-2 was cultured at an average titer of 106.13 TCID50/mL and used to evaluate the developed quantification method. Copy number concentrations of the cultured SARS-CoV-2 sgRNA and genomic RNA (gRNA) gave excellent linearity (R2 = 0.9999) with SARS-CoV-2 titers in the range from 500 to 105 TCID50/mL. Validation of 63 positive clinical samples further proves that the quantification of sgRNA-N by RT-dPCR is more sensitive for active virus quantitative detection. It is notable that we can infer the active virus titer through quantification of SARS-CoV-2 sgRNA based on the linear relationship in a biosafety level 2 laboratory within 3 h. It can be used to timely and effectively identify infectious patients and reduce unnecessary isolation especially when a large number of COVID-19 infected people impose a burden on medical resources.

The effect of voice training interventions on patients with oropharyngeal dysphagia: a systematic review.

BACKGROUND: Voice training has been proposed as an intervention to improve swallowing function in patients with dysphagia. However, little is known about the effects of voice training on swallowing physiology. OBJECTIVES: This systematic review investigates the effect of voice training on the swallowing function of patients with oropharyngeal dysphagia and provides the theoretical basis for improving the swallowing function and life quality of patients with oropharyngeal dysphagia. DATA SOURCES: A systematic review using a narrative synthesis approach of all published studies was sought with no date restrictions. Five electronic databases (EMBASE, PubMed, CINAHL, Web of Science, and The Cochrane Library) were searched from inception to April 2022. STUDY SELECTION: Eight studies were included. Two researchers screened the literature according to inclusion and exclusion criteria, extracted data, and carried out quality control according to the Cochrane handbook5.1.0. Data were analyzed narratively and descriptively. CONCLUSIONS: In general, statistically significant positive therapy effects were found. Voice training improves the oral and pharyngeal stages of swallowing in patients with neurological causes of dysphagia, such as stroke, and in patients with non-neurological causes of dysphagia, such as head and neck cancer. However, the current literature is limited and further primary research is required to provide more evidence to support voice training intervention in dysphagia.  Future studies could  further refine the content of voice training interventions, increase the number of patients enrolled, assess the long-term effects of voice training interventions and add associated assessments of the quality of life after treatment.

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro-transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.

Interlaboratory assessment of quantification of SARS-CoV-2 RNA by reverse transcription digital PCR.

The pandemic of the novel coronavirus disease 2019 (COVID-19) has caused severe harm to the health of people all around the world. Molecular detection of the pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), played a crucial role in the control of the disease. Reverse transcription digital PCR (RT-dPCR) has been developed and used in the detection of SARS-CoV-2 RNA as an absolute quantification method. Here, an interlaboratory assessment of quantification of SARS-CoV-2 RNA was organized by the National Institute of Metrology, China (NIMC), using in vitro transcribed RNA samples, among ten laboratories on six different dPCR platforms. Copy number concentrations of three genes of SARS-CoV-2 were measured by all participants. Consistent results were obtained with dispersion within 2.2-fold and CV% below 23% among different dPCR platforms and laboratories, and Z' scores of all the reported results being satisfactory. Possible reasons for the dispersion included PCR assays, partition volume, and reverse transcription conditions. This study demonstrated the comparability and applicability of RT-dPCR method for quantification of SARS-CoV-2 RNA and showed the capability of the participating laboratories at SARS-CoV-2 test by RT-dPCR platform.

Subcellular localization and RNAs determine FUS architecture in different cellular compartments.

Mutations in Fused in sarcoma (FUS) gene cause a subset of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron degenerative disease. Wild-type FUS is largely localized in the nucleus, but mutant FUS accumulates in the cytoplasm and forms inclusions. It is unclear whether FUS depletion from the nucleus or FUS inclusions in the cytoplasm triggers motor neuron degeneration. In this study, we revealed that the nuclear and cytoplasmic FUS proteins form distinct local distribution patterns. The nuclear FUS forms oligomers and appears granular under confocal microscope. In contrast, the cytoplasmic FUS forms inclusions with no oligomers detected. These patterns are determined by the subcellular localization of FUS, regardless of wild-type or mutant protein. Moreover, mutant FUS remained or re-directed in the nucleus can oligomerize and behave similarly to the wild-type FUS protein. We further found that nuclear RNAs are critical to its oligomerization. Interestingly, the formation of cytoplasmic FUS inclusions is also dependent on RNA binding. Since the ALS mutations disrupt the nuclear localization sequence, mutant FUS is likely retained in the cytoplasm after translation and interacts with cytoplasmic RNAs. We therefore propose that local RNA molecules interacting with the FUS protein in different subcellular compartments play a fundamental role in determining FUS protein architecture and function.

Evaluation of ascitic soluble human leukocyte antigen-G for distinguishing malignant ascites from benign ascites.

The overexpression of soluble human leukocyte antigen-G is associated with malignant tumours. The purpose of our study was to detect soluble human leukocyte antigen-G concentrations in ascites and to evaluate the value of ascitic soluble human leukocyte antigen-G for the diagnosis of malignant ascites. Enzyme-linked immunosorbent assay was used to detect soluble human leukocyte antigen-G levels in 64 patients with malignant ascites and 30 patients with benign ascites. Receiver operating characteristic curves were used to evaluate the diagnostic efficacy of ascitic soluble human leukocyte antigen-G for the detection of malignant ascites. Ascitic soluble human leukocyte antigen-G levels were significantly higher in the malignant ascites group than in the benign ascites group (20.718 ± 3.215 versus 12.467 ± 3.678 µg/L, t = 7.425, p < 0.001). The area under the receiver operating characteristic curve for ascitic soluble human leukocyte antigen-G was 0.957 (95% confidence interval, 0.872-0.992). At a cut-off value of 19.60 µg/L, the sensitivity and specificity of ascitic soluble human leukocyte antigen-G were 87.5% (95% confidence interval, 71.0%-96.5%) and 100% (95% confidence interval, 88.4%-100%), respectively. With respect to area under the receiver operating characteristic curve, sensitivity and specificity, ascitic carcinoembryonic antigen (0.810, 68.75% and 83.33%, respectively) and carbohydrate antigen 19-9 (0.710, 65.63% and 70%, respectively) significantly differed (all p < 0.05). In malignant ascites that were cytology-negative and biopsy-positive, the rate of positivity for ascitic soluble human leukocyte antigen-G was 75%, which was higher than the corresponding rates for ascitic carcinoembryonic antigen (31.25%) and carbohydrate antigen 19-9 (6.25%; both p < 0.05). In conclusion, The detection of ascitic soluble human leukocyte antigen-G exhibited good performance for diagnosing malignant ascites, and particularly those that were cytology-negative and biopsy-positive.

The RRM domain of human fused in sarcoma protein reveals a non-canonical nucleic acid binding site.

Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.

The prevalence of presbyphagia in older adults: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVE: Presbyphagia is defined as structural, physiological and innervational alterations in the swallowing process as a result of aging and is considered to be involved in the etiology of dysphagia. This systematic review and meta-analysis aimed to estimate the prevalence of presbyphagia in older adults without disease-related dysphagia. METHODS: In this study five databases were searched in October 2023 with no time limitation. Combined effect sizes of presbyphagia prevalence were calculated using random effect models. Meta-regression and subgroup analyses were conducted to identify sources of heterogeneity. Egger's test and a funnel plot were employed to examine publication bias. RESULTS: A total of 19 studies were selected for analysis. Overall, the prevalence of presbyphagia in older adults was 30.8% (95% confidence interval [CI] 24.8-36.7%). Publication bias was adjusted for using the fill-and-trim method and the corrected pooled prevalence of presbyphagia was 17.3% (95% CI 11.0-23.6%). In addition, the meta-regression findings revealed that the assessment tool had significant effects upon heterogeneity. CONCLUSION: Although the pooled prevalence of presbyphagia in older adults was 17.3%, the lack of large representative studies limited the interpretation of these findings. In the future, further large studies that diagnose presbyphagia using standardized assessment tools would facilitate new avenues to reduce the risk of dysphagia in older adults.

Proficiency testing for event-specific quantification of genetically modified maize MON87427: Performance assessment based on the metrologically traceable reference values as assigned values.

The Asia Pacific Metrology Program and the Accreditation Cooperation joint Proficiency Testing (PT) program for the quantification of genetically modified maize MON87427 was organized by the National Institute of Metrology, China, to enhance the measurement accuracy and metrological traceability in the region. Certified reference materials were employed as test samples; metrologically traceable certified reference values served as PT reference values (PTRVs) for evaluating the participants results. The consensus values obtained from the participants were higher than the assigned values, potentially due to the systematic effects of DNA extraction process. The participants' relatively poor overall performance by the ζ-score compared with z-score demonstrates their need to thoroughly investigate quantification bias to elevate the measurement capability of genetically modified (GM) content and deepen their understanding of uncertainty estimation. This program confirmed the importance of using metrologically traceable reference values instead of consensus values as PTRV for reliable performance assessment.

Identification of the metabolites after oral administration of extract of ziziphi spinosae semen to rats or dogs by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry.

Ziziphi spinosae semen (ZSS) is a traditional Chinese medicine that has been widely used to treat insomnia and anxiety. Modern pharmacological studies have demonstrated that flavonoids are the main active compounds in ZSS. However, the metabolites and the metabolic pathways of flavonoids in ZSS have not been investigated thoroughly. In this study, a method based on high-performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was established to identify the metabolites of flavonoids after oral administration of extract of ZSS to rats or dogs, using parent mass list-triggered data-dependent multiple-stage mass analysis at a resolving power of 50,000 in the external calibration mode. The mass accuracies obtained for all full-scan analyses were less than 4 ppm (<2 ppm in most cases). A total of 15 compounds were detected in biological samples of rats and dogs, and nine compounds were identified. The metabolic pathways of flavonoids of ZSS in rats and dogs were proposed. The results may help better understand the material basis and pharmacological mechanism of ZSS.

Portable Multi-Channel Electrochemical Device with Good Interaction and Wireless Connection for On-Site Testing.

It is very important to rapidly test the key indicators of water in the field to fully evaluate the quality of the regional water environment. However, a high-resolution measuring device that can generate small currents for low-concentration analytes in water samples is often bulky, complex to operate, and difficult for data sharing. This work introduces a portable multi-channel electrochemical device with a small volume, good interaction, and data-sharing capabilities called PMCED. The PMCED provides an easy-to-operate graphical interactive interface to conveniently set the parameters for cyclic voltammetry or a differential pulse method performed by the four electrode channels. At the same time, the device, with a current sensitivity of 100 nA V-1, was applied to the detection of water samples with high background current and achieved a high-resolution measurement at low current levels. The PMCED uses the Narrow Band Internet of Things (NB-IoT) to meet the needs for uploading data to the cloud in remote areas. The electrochemical signal preprocessing and chemometrics models run in the cloud, and the final results are visualized on a web page, providing a remote access channel for on-site testing results.

Relationship between SARS-CoV-2 nucleocapsid protein and N gene and its application in antigen testing kits evaluation.

More than forty antigen testing kits have been approved to response the prevalence of SARS-CoV-2 and its variant strains. However, the approved antigen testing kits are not capable of quantitative detection. Here, we successfully developed a lateral flow immunoassay based on colloidal gold nanoparticles (CGNP-based LFIA) for nucleocapsid (N) protein of SARS-CoV-2 quantitative detection. Delta strain (NMDC60042793) of SARS-CoV-2 have been cultured and analyzed by our developed digital PCR and LFIA methods to explore the relationship between N protein amount and N gene level. It indicated that the linear relationship (y = 47 ×) between N protein molecule number and N gene copy number exhibited very well (R2 = 0.995), the virus titers and N protein amount can be roughly estimated according to nucleic acid testing. Additionally, detection limits (LODs) of nine approved antigen testing kits also have been evaluated according to the Guidelines for the registration review of 2019-nCoV antigen testing reagents. Only three antigen testing kits had LODs as stated in the instructions, the LODs of Kits have been converted into the N gene and N protein levels, according to the established relationships among virus titer vers. N gene and antigen. Results demonstrated that the sensitivity of nucleic acid testing is at least 1835 times higher than that of antigen testing. We expect that the relationship investigation and testing kits evaluation have the important directive significance to precise epidemic prevention.

Fatty acid synthase expression and esophageal cancer.

Fatty acid synthase (FASN) overexpression has also been associated with a variety of human malignancies including tumor progression, aggressiveness, and metastasis. To investigate the role of FASN expression in esophageal cancer, we evaluated 60 cases of squamous cell carcinoma, 20 cases of adenocarcinoma, and 10 cases of normal esophageal tissues. We found that FASN was detected in 95 % human squamous cell carcinoma, and in 90 % human adenocarcinoma samples. However, all cases of normal esophageal epithelium did not express the protein of FASN. Further, to investigate the role of FASN in tumorigenesis and development, we analyze the growth and migration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation and wound healing assay. We found that inhibition of FASN expression in TE13 cells by RNAi suppressed the growth of cells. Decreased FASN expression mitigated the migration of TE13 cells. These studies demonstrated the functional importance of FASN in esophageal tumorigenesis, and suggested that inhibiting FASN might be applied to treat esophageal cancer.

Evaluation of a Most Probable Number Method for Detection and Quantification of Legionella pneumophila.

The detection and enumeration of Legionella pneumophila (L. pneumophila) in water is crucial for water quality management, human health and has been a research hotspot worldwide. Due to the time-consuming and complicated operation of the plate culture method, it is necessary to adopt a fast and effective method for application. The present study aimed to comprehensively evaluate the performance and applicability of the MPN method by comparing its qualitative and quantitative results with the GB/T 18204.5-2013 and ISO methods, respectively. The qualitative results showed that 372 samples (53%) were negative for both methods; 315 samples (45%) were positively determined by the MPN method, compared with 211 samples (30%) using GB/T 18204.5-2013. The difference in the detection rate between the two methods was statistically significant. In addition, the quantitative results showed that the concentration of L. pneumophila by the MPN method was greater than ISO 11731 and the difference was statistically significant. However, the two methods were different but highly correlated (r = 0.965, p < 0.001). The specificity and sensitivity of the MPN method were 89.85% and 95.73%, respectively. Overall, the results demonstrated that the MPN method has higher sensitivity, a simple operation process and good application prospects in the routine monitoring of L. pneumophila from water samples.

A droplet digital PCR assay for detection and quantification of Verticillium nonalfalfae and V. albo-atrum.

Verticillium nonalfalfae and V. albo-atrum are notorious pathogenic fungi that cause a destructive vascular disease called Verticillium wilt worldwide. Thus, timely and quantitative monitoring of fungal progression is highly desirable for early diagnosis and risk assessment. In this study, we developed a droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify V. nonalfalfae and V. albo-atrum. The performance of this assay was validated in comparison with that of a quantitative real-time polymerase chain reaction (qPCR) assay. The standard curve analysis of the ddPCR assay showed good linearity. The ddPCR assay indicated similar detection sensitivity to that of qPCR on pure genomic DNA, while it enhanced the positive rate for low-abundance fungi, especially in alfalfa stems. Receiver operating characteristic analysis revealed that ddPCR provided superior diagnostic performance on field tissues compared to qPCR, and the area under curve values were 0.94 and 0.90 for alfalfa roots and stems, respectively. Additionally, the quantitative results of the two methods were highly concordant (roots: R2 = 0.91; stems: R2 = 0.76); however, the concentrations determined by ddPCR were generally higher than those determined by qPCR. This discrepancy was potentially caused by differing amplification efficiencies for qPCR between cultured and field samples. Furthermore, the ddPCR assays appreciably improved quantitative precision, as reflected by lower coefficients of variation. Overall, the ddPCR method enables sensitive detection and accurate quantification of V. nonalfalfae and V. albo-atrum, providing a valuable tool for evaluating disease progression and enacting effective disease control.