Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus

[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.

Delayed organising pneumonia in an immunocompromised host after a mild COVID-19 infection.

Organising pneumonia after a mild COVID-19 infection has been increasingly reported and poses a diagnostic challenge to physicians especially in immunocompromised patients. We report a patient with a background of lymphoma in remission on rituximab who presented with prolonged and persistent fever after recovering from a mild COVID-19 infection. The initial workup showed bilateral lower zone lung consolidation; however, the infective and autoimmune workup were unremarkable. Subsequently, a bronchoscopy with transbronchial lung biopsy confirmed the diagnosis of organising pneumonia. A tapering glucocorticoid regimen was commenced with prompt resolution of the patient's clinical symptoms, and subsequent resolution of biochemical markers and radiological lung changes 3 months later. This case highlights the importance of early recognition of the diagnosis of organising pneumonia in immunocompromised populations after a mild COVID-19 infection as it shows promising response to glucocorticoid therapy.

Determinants of quality of life in frontline healthcare providers caring for COVID-19 patients.

AIMS AND OBJECTIVES: To investigate the factors affecting quality of life in healthcare providers who care for patients with COVID-19. BACKGROUND: Healthcare providers caring for COVID-19 patients during the pandemic suffered a deterioration in their quality of life. Several studies have explored their psychological impact of working with COVID patients, but none have examined the causes of this deterioration. DESIGN: A cross-sectional study. METHODS: In the current study, the authors investigated the factors affecting quality of life in 293 healthcare providers recruited from a medical centre in northern Taiwan who had recently cared for patients with suspected or confirmed COVID-19 by analysing their responses to an online self-report questionnaire, using bivariate correlations and structural equation modelling. Reporting of this research adheres to the STROBE guideline. RESULTS: The study identified an important sequence of factors that mediated the effects of perceived success of epidemic prevention policies, family relations problems and education level on quality of life in a sample of healthcare workers caring for COVID-19 patients. The mediators were use of approach-oriented coping strategies and current mental health status. Specifically, use of approach-oriented coping strategies was found to directly cause improved quality of life and indirectly cause improved mental health, whereas use of avoidant coping strategies was found to directly cause worsening of mental health. Poor mental health predicted poor quality of life. CONCLUSIONS: Results suggest that implementation of sound epidemic prevention policies that promote adoption of approach-oriented coping behaviour should lead to a better quality of life in the future for healthcare providers working in challenging circumstances. RELEVANCE TO CLINICAL PRACTICE: Assessment of these policies as well as the providers' family relations are necessary first steps to improving the success of approach-oriented coping behaviour in this population, which in turn can improve their mental health and quality of life. PATIENT OR PUBLIC CONTRIBUTION: Neither patients nor members of the public were involved in the design or execution of the study.

The role of the COVID-19 pandemic in time-frequency connectedness between oil market shocks and green bond markets: Evidence from the wavelet-based quantile approaches.

This study contributes to the existing literature on the relationship between oil market shocks and the green bond market by investigating the impact of the COVID-19 pandemic on their dynamic correlation. We first decompose the oil market shocks into components using a time-frequency framework. Then, we combine wavelet decomposition and quantile coherence and causality methods to discuss changes during the COVID-19 era. We observe positive effects of both supply-driven and demand-driven oil shocks on the green bond market at most quantile levels. However, supply-driven oil price changes play a major role. The results also indicate that long-term changes have a greater impact than short-term changes on the connection between oil and green bond markets. Nevertheless, the COVID-19 pandemic changed the nature of the causal relationship, as we observed no relationship under extreme market conditions during the pandemic era. We argue that the economic and social impacts of the COVID-19 pandemic have left investors focusing on the short-term substitution between oil and green bond markets.

Full-Thickness Macular Hole After Intravitreal Conbercept Injection in Branch Retinal Vein Occlusion.

This article reports a case of macular hole (MH) formation following intravitreal conbercept injection for branch retinal vein occlusion (BRVO). A 70-year-old male received three consecutive intravitreal injections of conbercept for the treatment of macular edema secondary to BRVO in his left eye. Due to the outbreak of the COVID-19 epidemic, the patient was lost to follow-up. At two months follow-up, a full-thickness MH was detected by fundoscopic and optical coherence tomography examination. Fortunately, the MH was successfully closed after pars plana vitrectomy. MH is a rare complication following intravitreal injections for RVO, which should be considered by clinicians.

Investigation of Neurological Complications after COVID-19 Vaccination: Report of the Clinical Scenarios and Review of the Literature.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), broke out in 2019 and became a pandemic in 2020. Since then, vaccines have been approved to prevent severe illness. However, vaccines are associated with the risk of neurological complications ranging from mild to severe. Severe complications such as vaccine-induced immune thrombotic thrombocytopenia (VITT) associated with acute ischaemic stroke have been reported as rare complications post-COVID-19 vaccination. During the pandemic era, VITT evaluation is needed in cases with a history of vaccination within the last month prior to the event. Cerebral venous sinus thrombosis (CVST) should be suspected in patients following immunization with persistent headaches who are unresponsive to analgesics. In this article, we investigated neurological complications after COVID-19 vaccination and provided more subsequent related clinical studies of accurate diagnosis, pathophysiological mechanisms, incidence, outcome, and management.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms.

We herein present an overview of the upcoming 5th edition of the World Health Organization Classification of Haematolymphoid Tumours focussing on lymphoid neoplasms. Myeloid and histiocytic neoplasms will be presented in a separate accompanying article. Besides listing the entities of the classification, we highlight and explain changes from the revised 4th edition. These include reorganization of entities by a hierarchical system as is adopted throughout the 5th edition of the WHO classification of tumours of all organ systems, modification of nomenclature for some entities, revision of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities, as well as inclusion of tumour-like lesions, mesenchymal lesions specific to lymph node and spleen, and germline predisposition syndromes associated with the lymphoid neoplasms.

CapsCovNet: A Modified Capsule Network to Diagnose COVID-19 From Multimodal Medical Imaging.

Since the end of 2019, novel coronavirus disease (COVID-19) has brought about a plethora of unforeseen changes to the world as we know it. Despite our ceaseless fight against it, COVID-19 has claimed millions of lives, and the death toll exacerbated due to its extremely contagious and fast-spreading nature. To control the spread of this highly contagious disease, a rapid and accurate diagnosis can play a very crucial part. Motivated by this context, a parallelly concatenated convolutional block-based capsule network is proposed in this article as an efficient tool to diagnose the COVID-19 patients from multimodal medical images. Concatenation of deep convolutional blocks of different filter sizes allows us to integrate discriminative spatial features by simultaneously changing the receptive field and enhances the scalability of the model. Moreover, concatenation of capsule layers strengthens the model to learn more complex representation by presenting the information in a fine to coarser manner. The proposed model is evaluated on three benchmark datasets, in which two of them are chest radiograph datasets and the rest is an ultrasound imaging dataset. The architecture that we have proposed through extensive analysis and reasoning achieved outstanding performance in COVID-19 detection task, which signifies the potentiality of the proposed model.

Phylogenetic and Spatiotemporal Analyses of the Complete Genome Sequences of Avian Coronavirus Infectious Bronchitis Virus in China During 1985-2020: Revealing Coexistence of Multiple Transmission Chains and the Origin of LX4-Type Virus.

Infectious bronchitis (IB) virus (IBV) causes considerable economic losses to poultry production. The data on transmission dynamics of IBV in China are limited. The complete genome sequences of 212 IBV isolates in China during 1985-2020 were analyzed as well as the characteristics of the phylogenetic tree, recombination events, dN/dS ratios, temporal dynamics, and phylogeographic relationships. The LX4 type (GI-19) was found to have the highest dN/dS ratios and has been the most dominant genotype since 1999, and the Taiwan-I type (GI-7) and New type (GVI-1) showed an increasing trend. A total of 59 recombinants were identified, multiple recombination events between the field and vaccine strains were found in 24 isolates, and the 4/91-type (GI-13) isolates were found to be more prone to being involved in the recombination. Bayesian phylogeographic analyses indicated that the Chinese IBVs originated from Liaoning province in the early 1900s. The LX4-type viruses were traced back to Liaoning province in the late 1950s and had multiple transmission routes in China and two major transmission routes in the world. Viral phylogeography identified three spread regions for IBVs (including LX4 type) in China: Northeastern China (Heilongjiang, Liaoning, and Jilin), north and central China (Beijing, Hebei, Shanxi, Shandong, and Jiangsu), and Southern China (Guangxi and Guangdong). Shandong has been the epidemiological center of IBVs (including LX4 type) in China. Overall, our study highlighted the reasons why the LX4-type viruses had become the dominant genotype and its origin and transmission routes, providing more targeted strategies for the prevention and control of IB in China.

The influence of the COVID-19 pandemic on the demand for different shades of green.

COVID-19 has heightened the dependence of urban dwellers on cultural ecosystem services provided by urban green spaces (UGS), specifically in regard to the provision of recreational opportunities, and psychological and physical health benefits arising from their use.As different types and levels of cultural ecosystem services are provided by different types of UGS, people may seek out different UGS to satisfy personal needs over various phases of COVID-19 mobility restrictions imposed by cities. We report on a study that took advantage of the different phases of COVID-19 mobility restrictions to assess the demand for and perception of different types of UGS in Singapore.The study utilised four datasets to compare demand for and visitorship patterns of UGS before the pandemic (Pre-Circuit Breaker), the duration of the strictest mobility restrictions (Circuit Breaker), and after the measures were relaxed (Post-Circuit Breaker). We used Google Search trends as a proxy for UGS demand, Google mobility data for an overview of population visitorship trends, visitor counts for granular insights on actual visitorship trends, and qualitative data on perception of parks by park visitors after restrictions eased. Parks were categorised as manicured and less manicured UGS for analysis.Search interest for UGS overall fell by more than 50% from during Circuit Breaker but the post-Circuit Breaker levels exceeded pre-Circuit Breaker, with a 70.9% increase for less manicured UGS compared to 20.8% for manicured UGS. This corroborated with Google mobility and visitor counts, which showed a steep decrease in park use followed by a rapid increase in the same periods, and with increased visitorship in the less manicured UGS. The perception study also showed that more than 50% of respondents reported visiting parks that they have never visited before, and there was a greater appreciation and use of UGS after the pandemic and preference for less manicured and more naturalistic landscapes.The pandemic has heightened the demand for cultural ecosystem services provided by UGS. Our study showed that this demand is not uniform across different types of UGS, with an increase visitorship and preference for less manicured green spaces. Read the free Plain Language Summary for this article on the Journal blog.

An acceptable methodological evaluation method of real-time fluorescent RT-PCR targeting SARS-CoV-2 (preprint)

Background: it is necessary to evaluate real-time fluorescent reverse-transcription PCR (RT-PCR) methods to detect the nucleic acids of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: considering lack of positive specimens in some particular locations in China, the specimens from healthy individuals were used to perform the methodology evaluations, in which the indexes were the differences of quantification cycle values (ΔCq) between human-derived internal reference control (IRC) genes of a specimen and quality control gene (QC). A series of experiments was conducted to evaluate various factors that might affect the results, such as types of virus transport media, methods of specimen pretreatment and template preparation, specimen vortex strength, specimen storage temperature, and duration. Results: it was better to store specimens in normal saline (NS) transport medium, release more virus particles from swabs by vortex mixing, extract nucleic acids with centrifugation methods, and perform amplification assays timely. The above-mentioned options and optimum conditions were further confirmed using SARS-CoV-2 pseudoviruses and positive clinical specimens. Conclusions: this study provides a solution for the accurate detection of SARS-CoV-2. Specifically, this study also indicates that the routine specimens from healthy individuals could be used to methodological evaluation of real-time fluorescent RT-PCR targeting SARS-CoV-2, of which the indexes were the ΔCq values.

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: In total, 605 serum samples from 125 COVID-19 patients (from January 1 to March 14, 2020) varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2. RESULTS: NAbs were detectable approximately 10 days post-onset (dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective. CONCLUSION: We demonstrated that NAb levels vary among all categories of COVID-19 patients. Long-term studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

One-year outcomes of patients with ST-segment elevation myocardial infarction during the COVID-19 pandemic.

The pandemic has led to adverse short-term outcomes for patients with ST-segment elevation myocardial infarction (STEMI). It is unknown if this translates to poorer long-term outcomes. In Singapore, the escalation of the outbreak response on February 7, 2020 demanded adaptation of STEMI care to stringent infection control measures. A total of 321 patients presenting with STEMI and undergoing primary percutaneous coronary intervention at a tertiary hospital were enrolled and followed up over 1-year. They were allocated into three groups based on admission date-(1) Before outbreak response (BOR): December 1, 2019-February 6, 2020, (2) During outbreak response (DOR): February 7-March 31, 2020, and (3) control group: November 1-December 31, 2018. The incidence of cardiac-related mortality, cardiac-related readmissions, and recurrent coronary events were examined. Although in-hospital outcomes were worse in BOR and DOR groups compared to the control group, there were no differences in the 1-year cardiac-related mortality (BOR 8.7%, DOR 7.1%, control 4.8%, p = 0.563), cardiac-related readmissions (BOR 15.1%, DOR 11.6%, control 12.0%, p = 0.693), and recurrent coronary events (BOR 3.2%, DOR 1.8%, control 1.2%, p = 0.596). There were higher rates of additional PCI during the index admission in DOR, compared to BOR and control groups (p = 0.027). While patients admitted for STEMI during the pandemic may have poorer in-hospital outcomes, their long-term outcomes remain comparable to the pre-pandemic era.

Privacy Perils of Open Data and Data Sharing: A Case Study of Taiwan's Open Data Policy and Practices

Governments and private sector players have hopped on the open data train in the past few years. Both the governments and civil society in Taiwan are exploring the opportunities provided by the data stored in public and private sectors. While they have been enjoying the benefits of the sharing and flowing of data among various databases, the government and some players in the private sectors have also posed tremendous privacy challenges by inappropriately gathering and processing personal data. The amended Personal Data Protection Act was originally enacted as a regulatory mechanism to protect personal data and create economic benefits via enhancing the uses of public and private sector data. In reality, the Act has instead resulted in harm to Taiwan's data privacy situation in this big data era. This article begins with an overview of the Taiwan's open data policy history and its current practices. Next, the article analyzes cases in which the data sharing practices between different sectors have given rise to privacy controversies, with a particular focus on 2020, when Taiwan used data surveillance in response to the COVID-19 pandemic. Finally, this article flags problems related to an open data system, including the protection of sensitive data, de-identification, the right to consent and opt-out, and the ambiguity of "public interest," and concludes by proposing a feasible architecture for the implementation of a more sensible open data system with privacy-enhancing characteristics. [ABSTRACT FROM AUTHOR] Copyright of Washington International Law Journal is the property of Pacific Rim Law & Policy Association and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Non-intrusive wastewater surveillance for monitoring of a residential building for COVID-19 cases.

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/respiratory illness). However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing.

The interval between onset and admission predicts disease progression in COVID-19 patients.

BACKGROUND: The prognostic role of the interval between disease onset and hospital admission (O-A interval) was undetermined in patients with the coronavirus disease 2019 (COVID-19). METHODS: A total of 205 laboratory-confirmed inpatients admitted to Hankou hospital of Wuhan from January 11 to March 8, 2020 were consecutively included in this retrospective observational study. Demographic data, medical history, laboratory testing results were collected from medical records. Univariate and multivariate logistic regression models were used to evaluate the prognostic effect of the O-A interval (≤7 versus >7 days) on disease progression in mild-to-moderate patients. For severe-to-critical patients, the in-hospital mortality and the length of hospital stay were compared between the O-A interval subgroups using log-rank test and Mann-Whitney U test, respectively. RESULTS: Mild-to-moderate patients with a short O-A interval (≤7 days) are more likely to deteriorate to severe-to-critical stage compared to those with a long O-A interval (>7 days) [unadjusted odds ratio =2.93, 95% confidence interval (CI), 1.32-6.55; adjusted odds ratio =3.44, 95% CI, 1.20-9.83]. No association was identified between the O-A interval and the mortality or the length of hospital stay of severe-to-critical patients. CONCLUSIONS: The O-A interval has predictive values for the disease progression in mild-to-moderate COVID-19 patients. Under circumstances of the specific health system in Wuhan, China, the spontaneous healthcare-seeking behavior is usually determined by patients' own heath conditions. Hence, the O-A interval can be reflective of the natural course of COVID-19 to some extent. However, our findings should be validated further in other cohorts and in other health systems.

Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4+ and CD8+ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

Impact of the COVID-19 Pandemic on Door-to-Balloon Time for Primary Percutaneous Coronary Intervention　- Results From the Singapore Western STEMI Network.

BACKGROUND: Little is known about the effect of the coronavirus disease 2019 (COVID-19) pandemic and the outbreak response measures on door-to-balloon time (D2B). This study examined both D2B and clinical outcomes of patients with STEMI undergoing primary percutaneous coronary intervention (PPCI).Methods and Results:This was a retrospective study of 303 STEMI patients who presented directly or were transferred to a tertiary hospital in Singapore for PPCI from October 2019 to March 2020. We compared the clinical outcomes of patients admitted before (BOR) and during (DOR) the COVID-19 outbreak response. The study outcomes were in-hospital death, D2B, cardiogenic shock and 30-day readmission. For direct presentations, fewer patients in the DOR group achieved D2B time <90 min compared with the BOR group (71.4% vs. 80.9%, P=0.042). This was more apparent after exclusion of non-system delay cases (DOR 81.6% vs. BOR 95.9%, P=0.006). Prevalence of both out-of-hospital cardiac arrest (9.5% vs. 1.9%, P=0.003) and acute mitral regurgitation (31.6% vs. 17.5%, P=0.006) was higher in the DOR group. Mortality was similar between groups. Multivariable regression showed that longer D2B time was an independent predictor of death (odds ratio 1.005, 95% confidence interval 1.000-1.011, P=0.029). CONCLUSIONS: The COVID-19 pandemic and the outbreak response have had an adverse effect on PPCI service efficiency. The study reinforces the need to focus efforts on shortening D2B time, while maintaining infection control measures.