Influence of waterborne butachlor (BUC), a commonly used pesticide, on morphometric, biochemical, and molecular biomarkers was evaluated in juvenile, full sibling, diploid and triploid African catfish (Clarias gariepinus). Fish were exposed for 21 days to one of three concentrations of BUC [mean measured µg/L: 22, 44 or 60]. Unexposed (control) triploids were heavier and longer and had higher visceral-somatic index (VSI) than diploids. Also, they had lighter liver weight (HSI) and showed lower transcript levels of brain gonadotropin-releasing hormone (GnRH), aromatase (cyp191b) and fushi tarazu-factor (ftz-f1), and plasma testosterone levels than diploids. Butachlor treatments had no effects, in either diploid or triploid fish, on VSI, HSI, weight or length changes, condition factor (CF), levels of plasma testosterone, 17-β estradiol (E2), cortisol, cholesterol, or mRNA levels of brain tryptophan hydroxylase (tph2), forkhead box L2 (foxl2), and 11 β-hydroxysteroid dehydrogenase type 2 (11β-hsd2). Expressions of cyp191b and ftz-f1 in triploids were upregulated by the two highest concentrations of BUC. In diploid fish, however, exposures to all BUC concentrations decreased GnRH transcription and the medium BUC concentration decreased ftz-f1 transcription. Substantial differences between ploidies in basal biomarker responses are consistent with the reported impaired reproductive axis in triploid C. gariepinus. Furthermore, the present study showed the low impact of short term exposure to BUC on reproductive axis in C. gariepinus.
Enzymatic synthesis of maltooligosaccharides is hampered due to lack of stability of soluble enzyme. This limitation can be tackled by cross linked enzyme aggregates (CLEAs) immobilization approach. However, substrate diffusion is a major bottleneck in cross linking technology. Herein, CLEAs of maltogenic amylase from Bacillus lehensis G1 (Mag1) was developed with addition of porous agent (Mag1-p-CLEAs). Comparison of thermal, pH and kinetic analysis with CLEAs without porous agent (Mag1-CLEAs) and free Mag1 was performed. Mag1-p-CLEAs with porous structure prepared at 0.8% (w/v) of citrus pectin (porous agent), 0.25% (w/v) of chitosan (cross linker) and cross linked for 1.5 h yielded 91.20% activity. 80% of activity is retained after 30 min of incubation at 40 °C and showed longer half-life than free Mag1 and Mag1-CLEAs. Mag1-p-CLEAs also showed pH stability at acidic and alkaline pH. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin accessibility. The increase in high catalytic efficiency (Kcat/Km) value, 1.90-fold and 1.05-fold showed that it also has better catalytic efficiency than free Mag1 and Mag1-CLEAs, respectively. Mag1-p-CLEAs not only improved substrate diffusibility of CLEAs, but also leads to higher thermal and pH stability of Mag1.
OBJECTIVES: Environmental risk factors contribute to 24% of the global burden of disease from all causes in disability-adjusted life years (DALYs), and to 23% of all global deaths. Malaysia being an advanced developing country, there is a need to prioritise environmental health issues to enable environmental health practitioners to focus on the most significant and urgent environmental health concerns.
CONTENT: This project was undertaken by a Thematic Working Group on Environmental Health Experts (TWG 10) under the Malaysian National Environmental Health Action Plan. Sixteen pre-selected environmental health issues were presented to a two focus group discussions among 20 environmental health and related professionals who then scored each issue on its magnitude and severity scale.
SUMMARY: The total of these scores generated a list of priority environmental health issues for Malaysia. Children environmental health came out as the environmental health issue of the highest priority.
OUTLOOK: We hope that this list of priority environmental health issues will be used for prioritising academic and professional manpower training, research funding allocation and planning for intervention programmes by various stakeholders.
Arsenic is a carcinogen element that occurs naturally in our environment. Humans can be exposed to arsenic through ingestion, inhalation, and dermal absorption. However, the most significant exposure pathway is via oral ingestion. Therefore, a comparative cross-sectional study was conducted to determine the local arsenic concentration in drinking water and hair. Then, the prevalence of arsenicosis was evaluated to assess the presence of the disease in the community. The study was conducted in two villages, namely Village AG and Village P, in Perak, Malaysia. Socio-demographic data, water consumption patterns, medical history, and signs and symptoms of arsenic poisoning were obtained using questionnaires. In addition, physical examinations by medical doctors were performed to confirm the signs reported by the respondents. A total of 395 drinking water samples and 639 hair samples were collected from both villages. The samples were analyzed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) to determine arsenic concentration. The results showed that 41% of water samples from Village AG contained arsenic concentrations of more than 0.01 mg/L. In contrast, none of the water samples from Village P exceeded this level. Whilst, for hair samples, 85 (13.5%) of total respondents had arsenic levels above 1 μg/g. A total of 18 respondents in Village AG had at least one sign of arsenicosis and hair arsenic levels of more than 1 μg/g. Factors significantly associated with increased arsenic levels in hair were female, increasing age, living in Village AG and smoking. The prevalence of arsenicosis in the exposed village indicates chronic arsenic exposure, and immediate mitigation action needs to be taken to ensure the wellbeing of the residents in the exposed village.
Sick building syndrome (SBS) is a collection of nonspecific syndromes linked with the built environment. The occurrence of SBS is associated with humidity, ventilation, moulds and microbial compounds exposure. However, no study has reported the association between indoor microbiome and SBS. In this study, 308 students were surveyed for SBS symptoms from 21 classrooms of 7 junior high schools from Johor Bahru, Malaysia, and vacuum dust from floor, desks and chairs was collected. High throughput amplicon sequencing (16S rRNA gene and ITS region) and quantitative PCR were conducted to characterize the absolute concentration of bacteria and fungi taxa. In total, 326 bacterial and 255 fungal genera were detected in dust with large compositional variation among classrooms. Also, half of these samples showed low compositional similarity to microbiome data deposited in the public database. The number of observed OTUs in Gammaproteobacteria was positively associated with SBS (p = 0.004). Eight microbial genera were associated with SBS (p
Tuberculosis (TB) cases have increased drastically over the last two decades and it remains as one of the deadliest infectious diseases in Malaysia. This cross-sectional study aimed to establish the spatial distribution of TB cases and its association with the sociodemographic and environmental factors in the Gombak district. The sociodemographic data of 3325 TB cases such as age, gender, race, nationality, country of origin, educational level, employment status, health care worker status, income status, residency, and smoking status from 1st January 2013 to 31st December 2017 in Gombak district were collected from the MyTB web and Tuberculosis Information System (TBIS) database at the Gombak District Health Office and Rawang Health Clinic. Environmental data consisting of air pollution such as air quality index (AQI), carbon monoxide (CO), nitrogen dioxide (NO2), sulphur dioxide (SO2), and particulate matter 10 (PM10,) were obtained from the Department of Environment Malaysia from 1st July 2012 to 31st December 2017; whereas weather data such as rainfall were obtained from the Department of Irrigation and Drainage Malaysia and relative humidity, temperature, wind speed, and atmospheric pressure were obtained from the Malaysia Meteorological Department in the same period. Global Moran's I, kernel density estimation, Getis-Ord Gi* statistics, and heat maps were applied to identify the spatial pattern of TB cases. Ordinary least squares (OLS) and geographically weighted regression (GWR) models were used to determine the spatial association of sociodemographic and environmental factors with the TB cases. Spatial autocorrelation analysis indicated that the cases was clustered (p<0.05) over the five-year period and year 2016 and 2017 while random pattern (p>0.05) was observed from year 2013 to 2015. Kernel density estimation identified the high-density regions while Getis-Ord Gi* statistics observed hotspot locations, whereby consistently located in the southwestern part of the study area. This could be attributed to the overcrowding of inmates in the Sungai Buloh prison located there. Sociodemographic factors such as gender, nationality, employment status, health care worker status, income status, residency, and smoking status as well as; environmental factors such as AQI (lag 1), CO (lag 2), NO2 (lag 2), SO2 (lag 1), PM10 (lag 5), rainfall (lag 2), relative humidity (lag 4), temperature (lag 2), wind speed (lag 4), and atmospheric pressure (lag 6) were associated with TB cases (p<0.05). The GWR model based on the environmental factors i.e. GWR2 was the best model to determine the spatial distribution of TB cases based on the highest R2 value i.e. 0.98. The maps of estimated local coefficients in GWR models confirmed that the effects of sociodemographic and environmental factors on TB cases spatially varied. This study highlighted the importance of spatial analysis to identify areas with a high TB burden based on its associated factors, which further helps in improving targeted surveillance.
Rhinitis is one of the most prevalent chronic diseases globally. Microbiome exposure affects the occurrence of rhinitis. However, previous studies did not differentiate allergic rhinitis (AR) and non-allergic rhinitis (NAR) in the microbial association analysis. In this study, we investigate 347 students in 8 junior high schools, Terengganu, Malaysia, who were categorized as healthy (70.9%), AR (13.8%) and NAR (15.3%) based on a self-administered questionnaire and skin prick tests of pollen, pet, mould and house dust mite allergens. Classroom microbial and metabolite exposure in vacuumed dust was characterized by PacBio long-read amplicon sequencing, quantitative PCR and LC-MS-based untargeted metabolomics. Our findings indicate a similar microbial association pattern between AR and NAR. The richness in Gammaproteobacteria was negatively associated with AR and NAR symptoms, whereas total fungal richness was positively associated with AR and NAR symptoms (p < 0.05). Brasilonema bromeliae and Aeromonas enteropelogenes were negatively associated with AR and NAR, and Deinococcus was positively associated with AR and NAR (p < 0.01). Pipecolic acid was protectively associated with AR and NAR symptoms (OR = 0.06 and 0.13, p = 0.009 and 0.045). A neural network analysis showed that B. bromeliae was co-occurring with pipecolic acid, suggesting that the protective role of this species may be mediated by releasing pipecolic acid. Indoor relative humidity and the weight of vacuum dust were associated with AR and NAR, respectively (p < 0.05), but the health effects were mediated by two protective bacterial species, Aliinostoc morphoplasticum and Ilumatobacter fluminis. Overall, our study reported a similar microbial association pattern between AR and NAR and also revealed the complex interactions between microbial species, environmental characteristics, and rhinitis symptoms.
Pathogens are commonly present in the human respiratory tract, but symptoms are varied among individuals. The interactions between pathogens, commensal microorganisms and host immune systems are important in shaping the susceptibility, development and severity of respiratory diseases. Compared to the extensive studies on the human microbiota, few studies reported the association between indoor microbiome exposure and respiratory infections. In this study, 308 students from 21 classrooms were randomly selected to survey the occurrence of respiratory infections in junior high schools of Johor Bahru, Malaysia. Vacuum dust was collected from the floor, chairs and desks of these classrooms, and high-throughput amplicon sequencing (16S rRNA and ITS) and quantitative PCR were conducted to characterize the absolute concentration of the indoor microorganisms. Fifteen bacterial genera in the classes Actinobacteria, Alphaproteobacteria, and Cyanobacteria were protectively associated with respiratory infections (p < 0.01), and these bacteria were mainly derived from the outdoor environment. Previous studies also reported that outdoor environmental bacteria were protectively associated with chronic respiratory diseases, such as asthma, but the genera identified were different between acute and chronic respiratory diseases. Four fungal genera from Ascomycota, including Devriesia, Endocarpon, Sarcinomyces and an unclassified genus from Herpotrichillaceae, were protectively associated with respiratory infections (p < 0.01). House dust mite (HDM) allergens and outdoor NO2 concentration were associated with respiratory infections and infection-related microorganisms. A causal mediation analysis revealed that the health effects of HDM and NO2 were partially or fully mediated by the indoor microorganisms. This is the first study to explore the association between environmental characteristics, microbiome exposure and respiratory infections in a public indoor environment, expanding our understanding of the complex interactions among these factors.
Little is known on respiratory effects of indoor chemicals in the tropics. We investigated associations between asthma and respiratory infections in Malaysian students and chemical exposure at home and at school. Moreover, we investigated differences in home environment between the three main ethnic groups in Malaysia (Malay, Chinese, Indian). Totally, 462 students from 8 junior high schools in Johor Bahru participated (96% participation rate). The students answered a questionnaire on health and home environment. Climate, carbon dioxide (CO2), volatile organic compounds (VOC), formaldehyde and nitrogen dioxide (NO2) were measured inside and outside the schools. Multilevel logistic regression was applied to study associations between exposure and health. Totally 4.8% were smokers, 10.3% had wheeze, 9.3% current asthma, and had 18.8% any respiratory infection in the past 3 months. Malay students had more dampness or mould (p
A prevalence study was conducted among office workers in Malaysia (N= 695). The aim of this study was to examine associations between asthma, airway symptoms, rhinitis and house dust mites (HDM) and cat allergy and HDM levels in office dust. Medical data was collected by a questionnaire. Skin prick tests were performed for HDM allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae) and cat allergen Felis domesticus. Indoor temperature and relative air humidity (RH) were measured in the offices and vacuumed dust samples were analyzed for HDM allergens. The prevalence of D. pteronyssinus, D. farinae and cat allergy were 50.3%, 49.0% and 25.5% respectively. Totally 9.6% had doctor-diagnosed asthma, 15.5% had current wheeze and 53.0% had current rhinitis. The Der p 1 (from D. pteronyssinus) and Der f 1 (from D. farinae) allergens levels in dust were 556 ng/g and 658 ng/g respectively. Statistical analysis was conducted by multilevel logistic regression, adjusting for age, gender, current smoking, HDM or cat allergy, home dampness and recent indoor painting at home. Office workers with HDM allergy had more wheeze (p= 0.035), any airway symptoms (p= 0.032), doctor-diagnosed asthma (p= 0.005), current asthma (p= 0.007), current rhinitis (p= 0.021) and rhinoconjuctivitis (p< 0.001). Cat allergy was associated with wheeze (p= 0.021), wheeze when not having a cold (p= 0.033), any airway symptoms (p= 0.034), doctor-diagnosed asthma (p= 0.010), current asthma (p= 0.020) and nasal allergy medication (p= 0.042). Der f 1 level in dust was associated with daytime breathlessness (p= 0.033) especially among those with HDM allergy. Der f 1 levels were correlated with indoor temperature (p< 0.001) and inversely correlated with RH (p< 0.001). In conclusion, HDM and cat allergies were common and independently associated with asthma, airway symptoms and rhinitis. Der f 1 allergen can be a risk factor for daytime breathlessness.
The status report on metal pollution in tropical estuaries and coastal waters is important to understand potential environmental health hazards. Detailed baseline measurements were made on physicochemical parameters (pH, temperature, redox potential, electrical conductivity, salinity, dissolved oxygen, total dissolved solid), major ions (Na, Ca, Mg, K, HCO3, Cl, SO4 and NO3) and metals concentrations ((27)Al, (75)As, (138)Ba, (9)Be, (111)Cd, (59)Co, (63)Cu, (52)Cr, (57)Fe, (55)Mn, (60)Ni, (208)Pb, (80)Se, (66)Zn) at estuaries and coastal waters along the Straits of Malacca. Principal component analysis (PCA) was employed to reveal potential pollution sources. Seven principal components were extracted with relation to pollution contribution from minerals-related parameters, natural and anthropogenic sources. The output from this study will generate a profound understanding on the metal pollution status and pollution risk of the estuaries and coastal system.
Rice is a carbohydrate, one of the plant-based foods that can accumulate heavy metal from soil and the irrigation water. Since total heavy metal always overestimates the amount of heavy metal available in rice, bioavailability of heavy metal is always preferred. Many studies have been done and found that in vitro methods offer an appealing alternative to human and animal studies. They can be simple, rapid, low in cost and may provide insights which not achievable in the in vivo studies. In vitro digestion model for rice may differ from other in vitro digestion models applied in soil or other type of foods studies. This review aims to provide an overview of in vitro digestion model used to determine bioavailability of heavy metal in rice, summarize health risk assessment application of heavy metal in rice studies and highlight the importance of health risk assessment to be included in the studies. Future exploration of in vitro digestion model and health risk assessment application on the bioavailability of heavy metal in rice was also suggested.
The objective of this article is to study the influence of exposure and socio-economic variables on the blood lead level of Malaysian school children. Data on respirable lead and blood lead of 346 school children were obtained from Kuala Lumpur (urban), Kemaman (semi-urban) and Setiu (rural). Respirable lead and blood lead were highest for Kuala Lumpur (95 ng/m3 and 5.26 micrograms/dL) followed by Kemaman (27 ng/m3 and 2.81 micrograms/dL) and Setiu (15 ng/m3 and 2.49 micrograms/dL), and the differences were statistically significant. The percentage of school children with excessive blood lead of 10 micrograms/dL or greater was 6.36% overall, and highest for Kuala Lumpur (11.73%). Regression analyses show that urban children are at higher risk of exhibiting excessive blood lead levels. Kuala Lumpur's school children have a 25 times greater risk of having excessive blood lead levels when compared to Kemaman's and Setiu's school children. Respirable and blood lead were correlated (r = 0.999, p = 0.021). Urban school children acquire higher blood lead levels than their rural and semi-urban counterparts, even after controlling for age, sex, parents' education and income levels. In conclusion, it is time that lead in the Malaysian environment and population be monitored closely, especially its temporal and spatial variability. Only then can a comprehensive preventive strategy be implemented.
COVID-19 pandemic is the greatest communicable disease outbreak to have hit Malaysia since the 1918 Spanish Flu which killed 34,644 people or 1% of the population of the then British Malaya. In 1999, the Nipah virus outbreak killed 105 Malaysians, while the SARS outbreak of 2003 claimed only 2 lives. The ongoing COVID-19 pandemic has so far claimed over 100 Malaysian lives. There were two waves of the COVID-19 cases in Malaysia. First wave of 22 cases occurred from January 25 to February 15 with no death and full recovery of all cases. The ongoing second wave, which commenced on February 27, presented cases in several clusters, the biggest of which was the Sri Petaling Tabligh cluster with an infection rate of 6.5%, and making up 47% of all cases in Malaysia. Subsequently, other clusters appeared from local mass gatherings and imported cases of Malaysians returning from overseas. Healthcare workers carry high risks of infection due to the daily exposure and management of COVID-19 in the hospitals. However, 70% of them were infected through community transmission and not while handling patients. In vulnerable groups, the incidence of COVID-19 cases was highest among the age group 55 to 64 years. In terms of fatalities, 63% were reported to be aged above 60 years, and 81% had chronic comorbidities such as diabetes, hypertension, and heart diseases. The predominant COVID-19 strain in Malaysia is strain B, which is found exclusively in East Asia. However, strain A, which is mostly found in the USA and Australia, and strain C in Europe were also present. To contain the epidemic, Malaysia implemented a Movement Control Order (MCO) beginning on March 18 in 4 phases over 2 months, ending on May 12. In terms of economic impacts, Malaysia lost RM2.4 billion a day during the MCO period, with an accumulated loss of RM63 billion up to the end of April. Since May 4, Malaysia has relaxed the MCO and opened up its economic sector to relieve its economic burden. Currently, the best approach to achieving herd immunity to COVID-19 is through vaccination rather than by acquiring it naturally. There are at least two candidate vaccines which have reached the final stage of human clinical trials. Malaysia's COVID-19 case fatality rate is lower than what it is globally; this is due to the successful implementation of early preparedness and planning, the public health and hospital system, comprehensive contact tracing, active case detection, and a strict enhanced MCO.
This is a cross-sectional study conducted among paddy farmers to characterize potential risk factors that influence levels of DNA damage from exposure to mixtures of organophosphates. Comet assay was used to determine the level of DNA damage by measuring the comet tail length from the exfoliated buccal mucosa. The result suggests that farmers who chronically exposure to a mixture of organophosphates has at least 2-fold significant increase of DNA damage as compared with control group. Factor analysis and linear regression both suggest that DNA damage reported by farmers may influence individual, occupational, and residential factors and are reported as significant predictor factors, whereas this effect is mainly caused by individual factors among the control group. The findings of the present study suggest that either farmer or control group bear certain extent of genotoxic burden contributed by different risk factors.
The Asia Pacific region is regarded as the most disaster-prone area of the world. Since 2000, 1.2 billion people have been exposed to hydrometeorological hazards alone through 1215 disaster events. The impacts of climate change on meteorological phenomena and environmental consequences are well documented. However, the impacts on health are more elusive. Nevertheless, climate change is believed to alter weather patterns on the regional scale, giving rise to extreme weather events. The impacts from extreme weather events are definitely more acute and traumatic in nature, leading to deaths and injuries, as well as debilitating and fatal communicable diseases. Extreme weather events include heat waves, cold waves, floods, droughts, hurricanes, tropical cyclones, heavy rain, and snowfalls. Globally, within the 20-year period from 1993 to 2012, more than 530 000 people died as a direct result of almost 15 000 extreme weather events, with losses of more than US$2.5 trillion in purchasing power parity.
Natural antioxidants derived from plants have shown a tremendous inhibitory effect on free radicals in actively metabolizing cells. Overproduction of free radicals increases the risk factor of chronic diseases associated with diabetes, cancer, arthritis and cardiovascular disease. Andrographis paniculata, Cinnamon zeylanicum, Curcuma xanthorrhiza, Eugenia polyantha and Orthosiphon stamineus are ethnomedicinal plants used in the Asian region to treat various illnesses from a common fever to metabolic disease. In this study, we have quantified the total phenolic (TPC) and flavonoid content (TFC) in these plants and its inhibitory effect on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals as well as the cytotoxicity effect on cell lines proliferation and zebrafish embryogenesis. Results showed that Cinnamon zeylanicum and E. polyantha have the highest phenolic and flavonoid content. Furthermore, both herbs significantly inhibited the formation of DPPH and ABTS free radicals. Meanwhile, O. stamineus exhibited minimum cytotoxicity and embryotoxicity on tested models. Good correlation between IC50 of 3T3-L1 cells and LC50 embyrotoxicity was also found. This study revealed the potent activity of antioxidant against free radical and the toxicology levels of the tested herbal plants.
In the last few decades, public health surveillance has increasingly applied statistical methods to analyze the spatial disease distributions. Nevertheless, contact tracing and follow up control measures for tuberculosis (TB) patients remain challenging because public health officers often lack the programming skills needed to utilize the software appropriately. This study aimed to develop a more user-friendly application by applying the CodeIgniter framework for server development, ArcGIS JavaScript for data display and a web application based on JavaScript and Hypertext Preprocessor to build the server's interface, while a webGIS technology was used for mapping. The performance of this approach was tested based on 3325 TB cases and their sociodemographic data, such as age, gender, race, nationality, country of origin, educational level, employment status, health care worker status, income status, residency status, and smoking status between 1st January 2013 and 31st December 2017 in Gombak, Selangor, Malaysia. These data were collected from the Gombak District Health Office and Rawang Health Clinic. Latitude and longitude of the location for each case was geocoded by uploading spatial data using Google Earth and the main output was an interactive map displaying location of each case. Filters are available for the selection of the various sociodemographic factors of interest. The application developed should assist public health experts to utilize spatial data for the surveillance purposes comprehensively as well as for the drafting of regulations aimed at to reducing mortality and morbidity and thus minimizing the public health impact of the disease.
BACKGROUND: This study aimed to provide an overview of lifestyle changes after breast cancer diagnosis and to examine the relationship between dietary and physical activity changes with weight changes in breast cancer patients. Women with breast carcinomas (n=368) were recruited from eight hospitals and four breast cancer support groups in peninsular Malaysia. Dietary and physical activity changes were measured from a year preceding breast cancer diagnosis to study entry. Mean duration since diagnosis was 4.86±3.46 years. Dietary changes showed that majority of the respondents had decreased their intake of high fat foods (18.8-65.5%), added fat foods (28.3-48.9%), low fat foods (46.8-80.7%), red meat (39.7%), pork and poultry (20.1-39.7%) and high sugar foods (42.1-60.9%) but increased their intake of fish (42.7%), fruits and vegetables (62.8%) and whole grains (28.5%). Intake of other food groups remained unchanged. Only a small percentage of the women (22.6%) had increased their physical activity since diagnosis where most of them (16.0%) had increased recreational activities. Age at diagnosis (β= -0.20, p= 0.001), and change in whole grain (β= -0.15, p= 0.003) and fish intakes (β= 0.13, p= 0.013) were associated with weight changes after breast cancer diagnosis. In summary, the majority of the women with breast cancer had changed their diets to a healthier one. However, many did not increase their physical activity levels which could improve their health and lower risk of breast cancer recurrence.