Evaluación de la vacuna multicomponente TcVac1 contra la enfermedad de Chagas utilizando un protocolo de electroporación intradérmica

Tesis de Doctorado de WAEL HEGAZY HASSAN MOUSTAFATrypanosoma cruzi (T. cruzi), un protozoario hemoflagelado, es el agente etiológico de la enfermedad de Chagas. Ésta es la causa más común de muertes relacionadas insuficiencia cardiaca congestiva entre los adultos jóvenes en las áreas endémicas de Sudamérica, Centroamérica y México. También se ha convertido en un problema de salud importante en los Estados Unidos y Europa debido a la migración a gran escala de latinoamericanos en las últimas décadas. Los esfuerzos para el desarrollo de vacunas contra la infección por T. cruzi han aumentado en los últimos años. Nuestro grupo de trabajo ha diseñado una serie de vacunas (TcVac) compuestas por antígenos de membrana anclados a la GPI derivados de T. cruzi. Se ha demostrado que las vacunas TcVac aumentan las respuestas inmunitarias humorales y celulares y proporcionan un control significativo (pero no completo) de la infección experimental en ratones y perros. En el presente documento, nos propusimos probar dos protocolos de inmunización para la entrega de la vacuna de ADN (TcVac1) compuesta de antígenos TcG2 y TcG4 en un modelo de ratón BALB/c. En un primer ensayo se comparó la protección conferida por la vacuna TcVac1 administrada vía intramuscular (IM) contra el protocolo de vacunación electroporación/intradérmica (IDE). Se vacunaron cuatro veces, con quince días de diferencia a doce ratones BALB/c por grupo. Dos semanas después de la última inmunización, se sacrificó a la mitad de los animales (n=6) de cada tratamiento, para la evaluación de la eficacia de la vacuna en la pre-infección y la segunda mitad (n=6) se sacrificaron 60 días después de la infección con trypomastigotes cepa Sylvio X10/4 de T. cruzi, para ser evaluados durante la fase aguda de la infección. La respuesta inmune humoral se evaluó mediante la evaluación de los subtipos de IgG anti-TcG2 y anti-TcG4 utilizando un ensayo de ELISA. La respuesta inmunitaria celular se evaluó mediante un ensayo de proliferación de linfocitos. Por último, se evaluaron los aspectos clínicos y morfopatológicos para todos los animales experimentales. Nuestros resultados demostraron que al comparar la entrega IDE TcVac1 frente a la entrega IM, el primero indujo un nivel significativamente mayor de respuesta de anticuerpos específicos por III antígeno (IgG2a+IgG2b>IgG1) y proliferación de linfocitos, que se expandió en respuesta al desafío de infección. La evaluación histológica después de la infección por desafío mostró infiltración de células inflamatorias (macrófagos y linfocitos) en el corazón y el tejido esquelético de todos los ratones infectados. Sin embargo, el mayor aumento de infiltrados inflamatorios se observó en ratones TcVac1_IDE/Tc en comparación con TcVac1_IM/Tc o ratones no vacunados/infectados. La extensión del infiltrado inflamatorio tisular se asoció directamente con el control de los nidos de amastigotes intracelulares en ratones vacunados/infectados (contra a los no vacunados/infectados). Nuestros resultados sugieren que la administración de IDE mejora la eficacia protectora de la vacuna TcVac1 contra la infección por T. cruzi en ratones en comparación con la administración IM de la vacuna. Posteriormente en un segundo experimento, se probó la eficacia de diferentes dosis vacunales utilizando la modificación a la vacuna eliminando los plásmidos que codifican para la interleucina-12 y factor estimulante de colonias granulocitos-macrófagos (GM-CSF). En este ensayo, se probaron cinco dosis diferentes (1, 5, 10, 15 y 20ug) de la vacuna para determinar la dosis óptima recomendada para prevenir la infección por T. cruzi con aplicación intradérmica/electroporación de la vacuna. En este experimento se utilizaron cuarenta y dos ratones hembra, cepa CD1, subdivididos en 7 tratamientos diferentes (n=6/grupo). Después de la vacunación, los ratones fueron desafiados con cepa Ninoa/MHOM/MX/1994 de T. cruzi (500 trypomastigotes/ratón, intraperitonealmente). Se analizaron parámetros tales como parasitemia, prueba de supervivencia, prueba serológica (ELISA) y análisis histopatológicos para evaluar la eficacia de la protección de la vacuna y determinar la dosis óptima. Los resultados demostraron que el grupo vacunado con 10 μg de dosis de ADN plasmídico indujeron los mejores niveles de inmunoglobulinas específicos, la parasitemia más baja y el mayor control del nidos de parásitos en cardiomiocitos y tejido muscular esquelético. Se confirmó que la aplicación intradérmica en conjunto con la electroporación es un método eficaz para la administración de vacunas con ADN, ya que mejora la inmunogenicidad del plásmido entregado, y reduce el tiempo y el costo de preparación de la vacuna, se minimiza la dosis de la vacuna, el número de IV refuerzos de la vacuna se reduce a una sola aplicación y la vacuna no requiere el uso de adyuvantes. La patología es una disciplina que depende crucialmente de la interpretación de imágenes para el diagnóstico y pronóstico de enfermedades. Tradicionalmente las técnicas de interpretación se basaron en gran medida en el análisis subjetivo de los especímenes, con un acuerdo variable entre los observadores. La interpretación moderna hace uso tanto del análisis molecular, como de la cuantificación objetiva de a través de microscopía auxiliada por sistemas de digitalización óptica. La dificultad para que el sistema ocular/cerebro humano analice objetivamente un objeto dentro de una escena, independientemente de su información contextual (por ejemplo, la percepción de un objeto dado puede ser muy diferente según su contexto), lo que promueve la necesidad de un ayuda microscopía computarizada para su uso en la patología molecular. De ahí que nuestro tercer experimento se enfocó en el desarrollo de un sistema digital de análisis de imágenes histopatológicas. En este tercer ensayo se utilizaron técnicas computarizadas de análisis digital de imágenes histopatológicas (técnicas de interpretación histológica basadas en el análisis de imágenes, cuantificación molecular de marcadores mixtos en estudios colorimétricos) enfocado en la detección y conteo de los infiltrados linfocitarios, conteo de cardiomiocitos y la determinación de hipertrofia cardiaca en ratones infectados con cepa Sylvio X10/4 de T. cruzi y la comparación de los resultados obtenidos con los resultados histológicos basados en observación y análisis visual realizado con tres patólogos en un ensayo doble ciego. Se usaron ratones BALB/c (n=48), los cuales fueron vacunados con TcVac1 vía intradérmica con electroporación, los ratones fueron sacrificados al día 60 post-infección y el análisis histopatológico se llevó a cabo como sigue; A) Se examinaron al menos cinco secciones por tejido (ventrículo derecho, ventrículo izquierdo y septum) para cada ratón para detectar la presencia de inflamación aguda y nidos de parásitos (magnificación: 400X y 100X), el parasitismo tisular se evaluó contando los nidos de amastigotes presentes en 100 campos microscópicos en cada uno de los corazones analizados. Del mismo modo, el V infiltrado inflamatorio se visualizó en >200 campos microscópicos de las secciones de tejido del corazón, tres patólogos analizaron en un estudio doble ciego las muestras, y se registraron los resultados finales del consenso. B) los tejidos fueron procesados en cortes 4 μm, teñidos con HE y analizados en las mismas secciones mencionados anteriormente. Los tejidos fueron observados bajo microscopio óptico y se llevó a cabo el análisis colorimétrico a través del software Image-Pro Plus 5.1. para todas las muestras. Finalmente, la comparación de los datos obtenidos de ambas técnicas fueron comparados. Los resultados mostraron la eficacia del análisis computarizado y la precisión de los datos obtenidos, donde había una diferencia estadísticamente significativa (P˂0.05) entre los grupos vacunados y no vacunados. la conclusión del presente experimento fue la obtención de datos cuantitativos para los estudios histopatológicos sin la necesidad de usar otras técnicas más costosas como inmunohistoquímica, FISH, ISH (silver-based), immunocitoquìmica, expresión de proteínas marcadas, etc. y evadir el margen de error humano en la recopilación y procesamiento de datos.UAEM con número de registro 3326/2012C, y al Consejo Nacional de Ciencia y Tecnología (CONACYT No. 156701), bajo la responsabilidad del Dr. Juan Carlos Vázquez Chagoyán Título: Correlación de las concentraciones séricas de troponina I con los cambios clínicos cardiovasculares en perros infectados con Trypanosoma cruzi en fase crónica Clave: 4518/2018/C

PROTECCION INMUNE CONTRA Trypanosoma cruzi INDUCIDO POR LA VACUNA TCVAC1 MODELO MURINO DE USANDO EL PROTOCOL DE ELECTROPORACIÓN INTRADÉRMICA

Trypanosoma cruzi, a parasitic protozoan, is the etiologic agent of Chagas disease. Chagas disease is the most common cause of congestive heart failure related deaths among young adults in the endemic areas of South and Central America and Mexico. It has also become an important health issue in the United States and Europe due to large scale migration of Latin Americans over the last few decades. No vaccines are currently available until now. In this study, we tested the vaccine efficacy of two antigen candidates against T. cruzi infection and disease in a mouse model. The use of TcVac1 (TcG2, TcG4, T cruzi antigen encoding plasmids, interleukin-12 [IL-12] and granulocyte-macrophage colony-stimulating factor [GMCSF] encoding plasmids as genetic adjuvants) anti T. cruzi candidate vaccine injected intramuscularly has been previously reported in mice with very encouraging results. Here we evaluated the comparative protection conferred by TcVac1 when administrated intramuscular (IM) versus an intradermal/electroporation (IDE) vaccination protocol. Twelve BALB/c mice per group were vaccinated four times fifteen days apart. Half the animals (n=6) from each treatment were sacrificed two weeks after the last immunization for pre-infection vaccine efficacy evaluation, and the second half (n=6) was sacrificed 60 days post-infection (dpi) with T.cruzi Trypomastigotes (Sylvio X10/4 strain). Immune response was assessed through anti-TcG2 and TcG4 T. cruzi antigens.TcVac1 induced a strong IgG response (IgG2b>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2b/IgG1 response in chronic phase. High IgG titers with IgG2 predominance in response to T. cruzi infection specific serum antibodies with an Enzyme Linked Immunosorbent Assay (ELISA) and lymphocyte activation against the studied antigens was evaluated through a lymphocyte proliferation assay. We found that IDE induced significantly larger surges of IgG antibodies including subtypes IgG1, IgG2a and IgG2b, during the pre- and post-infection periods for the two antigens used in the experiment. The ratio of antibodies IgG2b/IgG1 was >1 for TcG2 antigen in the pre-infection period in both administration routes. However for the TcG4 antigen the ratios were opposite for animals belonging to different administration routes1 for IM. During the post infection period for both treatments IgG2b/IgG1 ratio was always <1. Suggesting, as previously reported that a switch from Th1 to Th2 type immune response occurs in vaccinated/infected animals. During the Lymphocyte proliferation assays we observed that both antigens were able to induce lymphocyte proliferation during the pre-infection period. However, we observed that animals from the IDE group induced more proliferation than IM mice group when TcG4 was used to activate the cells, which was also observed during the post-infection phase of the experiment. No animals died due to infection, vaccinated mice appeared to have healthier status than the control animals

Genetic diversity of Bm86 sequences in Rhipicephalus (Boophilus) microplus ticks from Mexico: analysis of haplotype distribution patterns

Artículo científico derivado de Tesis doctoral de Saúl Gabriel Martínez ArzateBackground: Ticks are a problem for cattle production mainly in tropical and subtropical regions, because they generate great economic losses. Acaricides and vaccines have been used to try to keep tick populations under control. This has been proven difficult given the resistance to acaricides and vaccines observed in ticks. Resistance to protein rBm86-based vaccines has been associated with the genetic diversity of Bm86 among the ectoparasite’s populations. So far, neither genetic diversity, nor spatial distribution of circulating Bm86 haplotypes, have been studied within the Mexican territory. Here, we explored the genetic diversity of 125 Bm86 cDNA gene sequences from R. microplus from 10 endemic areas of Mexico by analyzing haplotype distribution patterns to help in understanding the population genetic structure of Mexican ticks. Results: Our results showed an average nucleotide identity among the Mexican isolates of 98.3%, ranging from 91.1 to 100%. Divergence between the Mexican and Yeerongpilly (the Bm86 reference vaccine antigen) sequences ranged from 3.1 to 7.4%. Based on the geographic distribution of Bm86 haplotypes in Mexico, our results suggest gene flow occurrence within different regions of the Mexican territory, and even the USA. Conclusions: The polymorphism of Bm86 found in the populations included in this study, could account for the poor efficacy of the current Bm86 antigen based commercial vaccine in many regions of Mexico. Our data may contribute towards designing new, highly-specific, Bm86 antigen vaccine candidates against R. microplus circulating in Mexico.SIEAE UAEM FES

Agreement of the Discrepancy Index Obtained Using Digital and Manual Techniques—A Comparative Study

The discrepancy index evaluates the complexity of the initial orthodontic diagnosis. The objective is to compare whether there is a difference in the final discrepancy index score of the American Board of Orthodontics (ABO) when obtained using digital and manual techniques. Fifty-six initial orthodontic records in a digital and physical format were included (28 each) in 2022 at the Center for Research and Advanced Studies in Dentistry. For the digital measurements, iTero and TRIOS 3 intraoral scanners were used, along with Insignia software and cephalometric tracing with Dolphin Imaging software. Manual measurements were obtained in dental casts using the ruler indicated for the previously mentioned discrepancy index, in addition to conventional cephalometric tracing. Student’s t-test did not show statistically significant differences between the digital and manual techniques, with final discrepancy index scores of 24.61 (13.34) and 24.86 (14.14), respectively (p = 0.769). Cohen’s kappa index showed very good agreement between both categorical measure ments (kappa value = 1.00, p = 0.001). The Bland–Altman method demonstrated a good agreement between continuous measurements obtained by both techniques with a bias of 0.2500 (superior limit of agreement =9.0092988, inferior limit of agreement = −8.5092988). Excellent agreement was observed in obtaining the discrepancy index through digital technique (Intraoral scanning and digital records) and manual technique (conventional records)

Effects of astaxanthin in mice acutely infected with Trypanosoma cruzi

During Trypanosoma cruzi infection, oxidative stress is considered a contributing factor for dilated cardiomyopathy development. In this study, the effects of astaxanthin (ASTX) were evaluated as an alternative drug treatment for Chagas disease in a mouse model during the acute infection phase, given its anti-inflammatory, immunomodulating, and anti-oxidative properties. ASTX was tested in vitro in parasites grown axenically and in co-culture with Vero cells. In vivo tests were performed in BALB/c mice (4–6 weeks old) infected with Trypanosoma cruzi and supplemented with ASTX (10 mg/kg/day) and/or nifurtimox (NFMX; 100 mg/kg/day). Results show that ASTX has some detrimental effects on axenically cultured parasites, but not when cultured with mammalian cell monolayers. In vivo, ASTX did not have any therapeutic value against acute Trypanosoma cruzi infection, used either alone or in combination with NFMX. Infected animals treated with NFMX or ASTX/NFMX survived the experimental period (60 days), while infected animals treated only with ASTX died before day 30 post-infection. ASTX did not show any effect on the control of parasitemia; however, it was associated with an increment in focal heart lymphoplasmacytic infiltration, a reduced number of amastigote nests in cardiac tissue, and less hyperplasic spleen follicles when compared to control groups. Unexpectedly, ASTX showed a negative effect in infected animals co-treated with NFMX. An increment in parasitemia duration was observed, possibly due to ASTX blocking of free radicals, an antiparasitic mechanism of NFMX. In conclusion, astaxanthin is not recommended during the acute phase of Chagas disease, either alone or in combination with nifurtimox.CONACYT PROY NO. 000000000156701. (REGISTRO INTERNO UAEM 3326

TcVac1 vaccine delivery by intradermal electroporation enhances vaccine induced immune protection against Trypanosoma cruzi infection in mice

Trabajo de investigación doctoral de Wael Hegazy Hassan Moustafa bajo la dirección de Juan Carlos Vázquez ChagoyánThe efforts for the development and testing of vaccines against Trypanosoma cruzi infection have increased during the past years. We have designed a TcVac series of vaccines composed of T. cruzi derived, GPI-anchored membrane antigens. The TcVac vaccines have been shown to elicit humoral and cellular mediated immune responses and provide significant (but not complete) control of experimental infection in mice and dogs. Herein, we aimed to test two immunization protocols for the delivery of DNA-prime/ DNA-boost vaccine (TcVac1) composed of TcG2 and TcG4 antigens in a BALB/c mouse model. Mice were immunized with TcVac1 through intradermal/electroporation (IDE) or intramuscular (IM) routes, challenged with T. cruzi, and evaluated during acute phase of infection. The humoral immune response was evaluated through the assessment of anti-TcG2 and anti-TcG4 IgG subtypes by using an ELISA. Cellular immune response was assessed through a lymphocyte proliferation assay. Finally, clinical and morphopathological aspects were evaluated for all experimental animals. Our results demonstrated that when comparing TcVac1 IDE delivery vs IM delivery, the former induced significantly higher level of antigen-specific antibody response (IgG2a + IgG2b > IgG1) and lymphocyte proliferation, which expanded in response to challenge infection. Histological evaluation after challenge infection showed infiltration of inflammatory cells (macrophages and lymphocytes) in the heart and skeletal tissue of all infected mice. However, the largest increase in inflammatory infiltrate was observed in TcVac1_IDE/Tc mice when compared with TcVac1_IM/Tc or non-vaccinated/infected mice. The extent of tissue inflammatory infiltrate was directly associated with the control of tissue amastigote nests in vaccinated/ infected (vs. non-vaccinated/infected) mice. Our results suggest that IDE delivery improves the protective efficacy of TcVac1 vaccine against T. cruzi infection in mice when compared with IM delivery of the vaccine.Universidad Autónoma de Estado de México (proyecto No. 3326/2012C), Consejo Nacional de Ciencia y Tecnología (Proyecto No. 156701) . Beca CONACyT a M.Sc. Wael Hegazy Hassan Moustafa (Beca numero No. 518232/291117)

Frequency and Distribution of Pulpal Calcifications in Teeth Involved in Jaw Tumors

Pulp calcifications are idiopathic mineralized masses associated with irritation, age, trauma, and systemic or genetic diseases. The objective of this work was to examine frequency and distribution of pulp calcifications in teeth involved in jaw tumors, analyzing their relationship with age, sex, location, size, and diagnosis of the lesion in a sample of 21 teeth associated with tumors of the jaws. Imaging analysis included CT scans, periapical X-rays, and orthopantomography of the clinical record; histological analysis included pulp tissue fixed in 10% buffered formalin for 24 h, with hematoxylin and eosin staining, and examined under light microscope. A chi-square test was applied to associate calcifications with all variables. The tumor lesions were from patients aged 17–66 years. Calcifications were observed in 38.1% of cases on image and histologically in 76.2%; 56% were nodular and 68.8% were distributed in chamber and root canal. The male sex presented a higher frequency of pulp calcifications, estimating a statistically significant difference with respect to women (p = 0.004); there was no statistical significance with the other variables. In conclusion, the pulp tissue of teeth affected by maxillary tumors presents a percentage of pulp calcifications similar to the tissue where the periradicular tissue is intact

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10&nbsp;years; 78.2% included were male with a median age of 37&nbsp;years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Global economic burden of unmet surgical need for appendicitis

Background: There is a substantial gap in provision of adequate surgical care in many low-and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods: Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results: Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion: For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially