Monitoring SARS-CoV-2 genetic variability: A post-market surveillance workflow for combined bioinformatic and laboratory evaluation of commercial RT-PCR assay performance.

OBJECTIVE: The speed at which Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is mutating has made it necessary to frequently assess how these genomic changes impact the performance of diagnostic real-time polymerase chain reaction (RT-PCR) assays. Herein, we describe a generic three-step workflow to assess the effect of genomic mutations on inclusivity and sensitivity of RT-PCR assays. METHODS: Sequences collected from the Global Initiative on Sharing All Influenza Data (GISAID) were mapped to a SARS-CoV-2 reference genome to evaluate the position and prevalence of mismatches in the oligonucleotide-binding sites of the QIAstat-Dx, an RT-PCR panel designed to detect SARS-CoV-2. The frequency of mutations and their impact on melting temperature were assessed, and sequences flagged by risk-based criteria were examined in vitro. RESULTS: Out of 8,900,393 SARS-CoV-2 genome sequences analyzed, only 173 (0.0019%) genomes contained potentially critical mutations for the QIAstat-Dx; follow-up in-vitro testing confirmed no impact on the assays' performance. CONCLUSIONS: The current study demonstrates that SARS-CoV-2 genetic variants do not affect the performance of the QIAstat-Dx device. It is recommended that manufacturers incorporate this workflow into obligatory post-marketing surveillance activities, as this approach could potentially enhance genetic monitoring of their product.

Flagella, Type I Fimbriae and Curli of Uropathogenic Escherichia coli Promote the Release of Proinflammatory Cytokines in a Coculture System.

BACKGROUND: Urinary tract infections (UTIs) are a public health problem in Mexico, and uropathogenic Escherichia coli (UPEC) is one of the main etiological agents. Flagella, type I fimbriae, and curli promote the ability of these bacteria to successfully colonize its host. AIM: This study aimed to determine whether flagella-, type I fimbriae-, and curli-expressing UPEC induces the release of proinflammatory cytokines in an established coculture system. METHODS: The fliC, fimH, and csgA genes by UPEC strain were disrupted by allelic replacement. Flagella, type I fimbriae, and curli were visualized by transmission electron microscopy (TEM). HTB-5 (upper chamber) and HMC-1 (lower chamber) cells cocultured in Transwell® plates were infected with these UPEC strains and purified proteins. There was adherence to HTB-5 cells treated with different UPEC strains and they were quantified as colony-forming units (CFU)/mL. RESULTS: High concentrations of IL-6 and IL-8 were induced by the FimH and FliC proteins; however, these cytokines were detected in low concentrations in presence of CsgA. Compared with UPEC CFT073, CFT073ΔfimH, CFT073ΔfimHΔfliC, and CFT073ΔcsgAΔfimH strains significantly reduced the adherence to HTB-5 cells. CONCLUSION: The FimH and FliC proteins are involved in IL-6 and IL-8 release in a coculture model of HTB-5 and HMC-1 cells.

[Prevalencia de complicaciones de la diabetes y comorbilidades asociadas en medicina familiar del Instituto Mexicano del Seguro Social].

Introduction: The prevalence of chronic complications and comorbidities in patients with type 2 diabetes (T2D) has increased worldwide. Objective: To compare the prevalence of complications and chronic comorbidities in patients with T2D at 36 family medicine units of five chapters of the Mexican Institute of Social Security (IMSS). Method: Complications (hypoglycemia, diabetic foot, kidney disease, retinopathy, ischemic heart disease, cerebrovascular disease and heart failure) and comorbidities (liver disease, cancer and anemia) were identified according to codes of the International Classification of Diseases, 10th Revision. Comparisons were made by chapter, age, gender and evolution time. Results: Complications and comorbidities were more common in subjects aged ≥ 62 years. Out of 297 100 patients, 34.9 % had any complication; microvascular complications (32 %) prevailed in the industrial North, whereas macrovascular complications (12.3 %) did in the rural East, and comorbidities (5 %) in southern Mexico City. Complications predominated in men (any complication, 30.2 %). Heart failure and comorbidities were more common in women (5.6 % and 4.9 %, respectively). Conclusions: T2D complications and comorbidities showed geographic and gender differences, and were greater with older age and longer evolution time. It is urgent for strategies for the prevention of complications and comorbidities to be reinforced in patients with T2D.

Introducción: La prevalencia de complicaciones crónicas y comorbilidades en pacientes con diabetes tipo 2 (DT2) se han incrementado en el mundo. Objetivo: Comparar la prevalencia de complicaciones y comorbilidades crónicas en pacientes con DT2 en 36 unidades de medicina familiar de cinco delegaciones del Instituto Mexicano del Seguro Social (IMSS). Métodos: Conforme los códigos de la Décima Revisión de la Clasificación Internacional de Enfermedades se identificaron las complicaciones (hipoglucemia, pie diabético, enfermedad renal, retinopatía, enfermedad cardiaca isquémica, enfermedad cerebrovascular y falla cardiaca) y comorbilidades (enfermedad hepática, cáncer, anemia) de DT2. Se compararon por delegación, edad, sexo y tiempo de evolución. Resultados: Las complicaciones y comorbilidades fueron más comunes en personas ≥ 62 años. De 297 100 pacientes, 34.9 % presentó cualquier complicación; microvasculares en el norte industrial (32 %), macrovasculares en el este rural (12.3 %) y comorbilidades (5 %) en el sur de la Ciudad de México; estas complicaciones predominaron en los hombres (cualquier complicación 30.2 %). La falla cardiaca y las comorbilidades fueron más comunes en mujeres (5.6 y 4.9 %). Conclusiones: Las complicaciones y comorbilidades de DT2 mostraron diferencias geográficas y de sexo y fueron mayores con la edad y el tiempo de evolución. Urge reforzar estrategias para la prevención de las complicaciones y comorbilidades en los pacientes con DT2.

Prevalencia de complicaciones de la diabetes y comorbilidades asociadas en medicina familiar del Instituto Mexicano del Seguro Social / Prevalence of complications of diabetes and associated comorbidities in family medicine of the Mexican Institute

Resumen Introducción: La prevalencia de complicaciones crónicas y comorbilidades en pacientes con diabetes tipo 2 (DT2) se han incrementado en el mundo. Objetivo: Comparar la prevalencia de complicaciones y comorbilidades crónicas en pacientes con DT2 en 36 unidades de medicina familiar de cinco delegaciones del Instituto Mexicano del Seguro Social (IMSS). Métodos: Conforme los códigos de la Décima Revisión de la Clasificación Internacional de Enfermedades se identificaron las complicaciones (hipoglucemia, pie diabético, enfermedad renal, retinopatía, enfermedad cardiaca isquémica, enfermedad cerebrovascular y falla cardiaca) y comorbilidades (enfermedad hepática, cáncer, anemia) de DT2. Se compararon por delegación, edad, sexo y tiempo de evolución. Resultados: Las complicaciones y comorbilidades fueron más comunes en personas ≥ 62 años. De 297 100 pacientes, 34.9 % presentó cualquier complicación; microvasculares en el norte industrial (32 %), macrovasculares en el este rural (12.3 %) y comorbilidades (5 %) en el sur de la Ciudad de México; estas complicaciones predominaron en los hombres (cualquier complicación 30.2 %). La falla cardiaca y las comorbilidades fueron más comunes en mujeres (5.6 y 4.9 %). Conclusiones: Las complicaciones y comorbilidades de DT2 mostraron diferencias geográficas y de sexo y fueron mayores con la edad y el tiempo de evolución. Urge reforzar estrategias para la prevención de las complicaciones y comorbilidades en los pacientes con DT2.

Abstract Introduction: The prevalence of chronic complications and comorbidities in patients with type 2 diabetes (T2D) has increased worldwide. Objective: To compare the prevalence of complications and chronic comorbidities in patients with T2D at 36 family medicine units of five chapters of the Mexican Institute of Social Security (IMSS). Method: Complications (hypoglycemia, diabetic foot, kidney disease, retinopathy, ischemic heart disease, cerebrovascular disease and heart failure) and comorbidities (liver disease, cancer and anemia) were identified according to codes of the International Classification of Diseases, 10th Revision. Comparisons were made by chapter, age, gender and evolution time. Results: Complications and comorbidities were more common in subjects aged ≥ 62 years. Out of 297 100 patients, 34.9 % had any complication; microvascular complications (32 %) prevailed in the industrial North, whereas macrovascular complications (12.3 %) did in the rural East, and comorbidities (5 %) in southern Mexico City. Complications predominated in men (any complication, 30.2 %). Heart failure and comorbidities were more common in women (5.6 % and 4.9 %, respectively). Conclusions: T2D complications and comorbidities showed geographic and gender differences, and were greater with older age and longer evolution time. It is urgent for strategies for the prevention of complications and comorbidities to be reinforced in patients with T2D.

Agreement between the 'point of care' tests for microalbuminuria and HbA1c performed in mexican family medicine units and the results of standard laboratory tests.

The albumin-creatinine ratio is considered an indicator of microalbuminuria, precursor to chronic kidney disease, while HbA1c is used to measure glycemic control. Given the prevalence of diabetes-related nephropathy, spot testing of albumin has long been recommended as a preventative measure, for the timely detection of microalbuminuria. However, many countries do not have this testing available in primary care, and sometimes not even in second- and third-level care. The objective of this study was to compare agreement of the microalbuminuria and HbA1c results obtained in the laboratory with 'gold standard' techniques, with those obtained on site with a 'Point of Care' DCA Vantage™ device by Siemens. Results for the albumin-creatinine ratio and HbA1c from the Siemens DCA Vantage™ point of care device were compared with those from standard laboratory tests in 25 family medicine units in Mexico City and Toluca, State of Mexico, in patients diagnosed with type-2 diabetes. Agreement between the albumin values of the 2 tests was 0.745 (CI 95% 0.655-0.812). Agreement between the two measurement techniques for HbA1c was 0.970 (CI 95% 0.966-0.973). The results obtained were sufficiently comparative (Ri= 0.74 for albumin-creatinine ratio and Ri = 0.97 for HbA1c) to justify the use of the point of care device. Given the high agreement between the point of care device and laboratory tests, this device could be used to identify chronic kidney disease and glycemic control for more adequate treatment in patients with diabetes, especially in remote areas.

Stepwise strategies to successfully recruit diabetes patients in a large research study in Mexican population.

AIMS: Describe stepwise strategies (electronic chart review, patient preselection, call-center, personnel dedicated to recruitment) for the successful recruitment of >5000 type 2 diabetes patients in four months. METHODS: Twenty-five family medicine clinics from Mexico City and the State of Mexico participated: 13 usual care, 6 specialized diabetes care and 6 chronic disease care. Appointments were scheduled from 11/3/2015 to 3/31/2016. Phone calls were generated automatically from an electronic database. A telephone questionnaire verified inclusion criteria, and scheduled an appointment, with a daily report of appointments, patient attendance, acceptance rate, and questionnaire completeness. Another recruitment log reviewed samples collected. Absolute number (percentage) of patients are reported. Means and standard deviations were estimated for continuous variables, χ2 test and independent "t" tests were used. OR and 95% CI were estimated. RESULTS: 14,358 appointments were scheduled, 9146 (63.7%) attended their appointment: 5710 (62.4%) fulfilled inclusion criteria and 5244 agreed to participate (91.8% acceptance). Those accepting participation were more likely women, younger and with longer disease duration (p<0.05). The cost of the call-center service was $3,010,000.00 Mexican pesos (∼$31.70 USD per recruited patient). CONCLUSIONS: Stepwise strategies recruit a high number of patients in a short time. Call centers offer a low cost per patient.

Population-based prevalence of cervical infection with human papillomavirus genotypes 16 and 18 and other high risk types in Tlaxcala, Mexico.

BACKGROUND: Cervical cancer remains an important cause of cancer mortality for Mexican women. HPV 16/18 typing may help to improve cervical cancer screening. Here we present the prevalence of high-risk human papillomavirus (hrHPV) including HPV16 and HPV18 from the FRIDA (Forwarding Research for Improved Detection and Access) population. METHODS: Beginning in 2013, we recruited 30,829 women aged 30-64 in Tlaxcala, Mexico. Cervical samples were collected and tested for 14 hrHPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). We used logistic regression to estimate odds ratios with 95 % confidence intervals for hrHPV infections according to putative risk factors. RESULTS: Prevalence of infection with any of the 14 hrHPV types was 11.0 %. The age-specific prevalence of all hrHPV formed a U-shaped curve with a higher prevalence for women aged 30-39 and 50-64 than women aged 40-49. Across all age groups, 2.0 % of women were positive for HPV16 and/or HPV18 (HPV16/18), respectively. HPV16/18 prevalence also showed a U-shaped curve with increased prevalence estimates for women aged both 30-39 and 60-64. Both prevalence curves had a significant quadratic age coefficient. Infections with hrHPV were positively associated with an increased number of lifetime sexual partners, a history of sexually transmitted disease, being unmarried, use of hormonal contraception, having a history of smoking and reported condom use in the multivariate model. CONCLUSIONS: The FRIDA population has a bimodal distribution of both hrHPV and HPV16/18 positivity with higher prevalences at ages 30-39 and 60-64. These findings will help to evaluate triage algorithms based on HPV genotyping. TRIAL REGISTRATION: The trial is registered with ClinicalTrials.gov, number NCT02510027 .

Triage strategies in cervical cancer detection in Mexico: methods of the FRIDA Study.

OBJECTIVE: This paper describes the study design and baseline characteristics of the study population, including the first 30 829 women who enrolled in the Forwarding Research for Improved Detection and Access for Cervical Cancer Screening and Triage (FRIDA Study). This is a large population based study that is evaluating the performance and cost-effectiveness of different triage strategies for high-risk HPV (hrHPV) positive women in Mexico. MATERIALS AND METHODS: The target population is more than 100 000 women aged 30 to 64 years who attend the Cervical Cancer Screening Program in 100 health centers in the state of Tlaxcala, Mexico. Since August 2013, all women in the region have been invited to enroll in the study. The study participants are evaluated to determine hrHPV infection using the Cobas 4800 HPV test. The HPV-16/18 genotyping and cytology triage strategies are performed as reflex tests in all hrHPV-positive participants. Women with a positive HPV-16/18 test and/or abnormal cytology (atypical squamous cells of undetermined significance or worse, ASCUS+) are referred for colposcopy evaluation, where a minimum of four biopsies and an endocervical sample are systematically collected. Histologic confirmation is performed by a standardized panel of pathologists. RESULTS: Among the 30 829 women who have been screened, the overall prevalence of hrHPV is 11.0%. The overall prevalence of HPV16 and HPV18 are 1.5% and 0.7%, respectively. Cytological abnormalities (ASCUS+) were detected in 11.8% of the hrHPV-positive women. A total of 27.0% (920/3,401) of the hrHPV-positive women were referred to colposcopy because of a positive HPV16/18 test and/or abnormal reflex cytology, (31.6% had only ASCUS+, 53.6% were HPV16/18 positive with a normal cytology result, and 9.5% were positive to both triage tests). CONCLUSION: The results of this study will help policy makers and health service providers establish the best practices for triage in cervical cancer screening in Mexico and other countries.

Triage strategies in cervical cancer detection in Mexico: methods of the FRIDA Study / Estrategias de triage en la detección de cáncer cervical: métodos del estudio FRIDA

Abstract Objective: This paper describes the study design and baseline characteristics of the study population, including the first 30 829 women who enrolled in the Forwarding Research for Improved Detection and Access for Cervical Cancer Screening and Triage (FRIDA Study). This is a large population based study that is evaluating the performance and cost-effectiveness of different triage strategies for high-risk HPV (hrHPV) positive women in Mexico. Materials and methods: The target population is more than 100 000 women aged 30 to 64 years who attend the Cervical Cancer Screening Program in 100 health centers in the state of Tlaxcala, Mexico. Since August 2013, all women in the region have been invited to enroll in the study. The study participants are evaluated to determine hrHPV infection using the Cobas 4800 HPV test. The HPV-16/18 genotyping and cytology triage strategies are performed as reflex tests in all hrHPV-positive participants. Women with a positive HPV-16/18 test and/or abnormal cytology (atypical squamous cells of undetermined significance or worse, ASCUS+) are referred for colposcopy evaluation, where a minimum of four biopsies and an endocervical sample are systematically collected. Histologic confirmation is performed by a standardized panel of pathologists. Results: Among the 30 829 women who have been screened, the overall prevalence of hrHPV is 11.0%. The overall prevalence of HPV16 and HPV18 are 1.5% and 0.7%, respectively. Cytological abnormalities (ASCUS+) were detected in 11.8% of the hrHPV-positive women. A total of 27.0% (920/3,401) of the hrHPV-positive women were referred to colposcopy because of a positive HPV16/18 test and/or abnormal reflex cytology, (31.6% had only ASCUS+, 53.6% were HPV16/18 positive with a normal cytology result, and 9.5% were positive to both triage tests). Conclusion: The results of this study will help policy makers and health service providers establish the best practices for triage in cervical cancer screening in Mexico and other countries.

Resumen Objetivo: El objetivo de este artículo es describir el diseño del estudio FRIDA y las características basales de las primeras 30 829 mujeres tamizadas. El estudio FRIDA (Forwarding Research for Improved Detection and Access for Cervical Cancer Screening and Triage) es un estudio de demostración con base poblacional diseñado para evaluar el desempeño y costo-efectividad de diferentes alternativas de triage en mujeres VPH de alto riesgo (VPHar) positivas bajo condiciones reales de un programa de tamizaje para cáncer cervical en México. Material y métodos: La población objetivo la conforman poco más de 100 000 mujeres de 30 a 64 años que asisten al programa de detección oportuna de cáncer cervical en alguno de los 100 centros de salud de la jurisdicción sanitaria 1 de Tlaxcala. Desde agosto de 2013, todas las mujeres son invitadas al estudio. Las participantes del estudio son tamizadas para determinar la infección con VPHar mediante la prueba VPHar Cobas 4800. Se realizan las pruebas de triage de tipificación de VPH16/18 y citología en todas las mujeres con resultados VPHar positivos. Las mujeres con un resultado positivo a VPH16/18 y/o citología anormal (células escamosas atípicas de resultado incierto o peor: ASCUS+) son referidas a evaluación colposcópica, seguida de una colección sistemática de un mínimo de cuatro biopsias cervicales y un cepillado endocervical. La confirmación histológica se lleva a cabo por un panel de patólogos. Resultados Un total de 30 829 mujeres han sido tamizadas, con una prevalencia de VPHar del 11.0%. La prevalencia global de VPH16 y VPH18 es 1.5% y 0.7%, respectivamente. Se detectó un 11.8% de anormalidades citológicas (ASCUS+). Entre las mujeres VPHar positivas, la prevalencia de un resultado de triage positivo (VPH16/18 o citología anormal) fue 27.0%, distribuido de la siguiente forma, 31.6% de éstos fueron sólo ASCUS+ VPH16/18 negativo, 53.6% fueron VPH 16/18 positivos y citología normal, y 9.5% positivos a ambas pruebas de triage. Conclusión: Los resultados de este estudio ayudarán tanto a los tomadores de decisiones como a los proveedores de servicios de salud a establecer la mejor estrategia de triage en programas de tamizaje de cáncer cervical basados en VPHar en México y en otros países.

Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel.

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.

O-glycosylation expression in fibroadenoma.

Fibroadenomas are human benign breast tumors characterized by proliferation of epithelial and stroma cells of the terminal ductal unit. Expression of O-glycans seems to contribute to the proliferation and transformation events. With this in mind, we evaluated the expression of glycans in fibroadenoma tissue through immunohistochemistry with antibodies against mucin epitopes (Anti CA15-3 and MUC1), as well as with lectins specific for glycans linked to proteins or lipids, and we compared findings with healthy breast specimens. Our results show positive expression of CA15-3 and MUC1 in fibroadenoma tissue, mainly in duct and stroma cells, whereas, in normal samples, staining was observed in duct cells. The lectin from Glycine max recognized equally well duct and stroma cells; this was the only lectin showing co-localization with anti-CA15-3 in healthy and tumor tissues. Dolichos biflorus, Artocarpus integrifolia, and Griffonia simplicifolia lectins recognized duct cells in control healthy tissues as well as in fibroadenoma tissue. The lectin from Amaranthus leucocarpus recognized only duct cells in control samples, whereas, in fibroadenoma tissue, it recognized duct and some stromal cells, suggesting that O-glycans-type mucin linked to proteins and mucin participate in the development of fibroadenomas.