Campylobacter spp. Prevalence in Santiago, Chile: A Study Based on Molecular Detection in Clinical Stool Samples from 2014 to 2019.

Campylobacter spp. is an emerging cause of infectious diarrhea worldwide. In South American countries such as Chile, its prevalence is underestimated due to inadequate detection methods. Gastrointestinal multiplex PCR panels (GMP) permit rapid and sensitive detection of bacterial pathogens and provide important epidemiological information. This study aimed to analyze Campylobacter epidemiology using the results of molecular methods and to compare molecular detection results to those of culture methods. We performed a retrospective, descriptive analysis of Campylobacter spp. detected in clinical stool samples between 2014-2019 by GMP and culture. Within 16,582 specimens examined by GMP, Campylobacter was the most prevalent enteropathogenic bacteria (8.5%), followed by Salmonella spp. (3.9%), Shigella spp./enteroinvasive Escherichia coli (EIEC) (1.9%), and Yersinia enterocolitica (0.8%). The highest Campylobacter prevalence occurred in 2014/2015. Campylobacteriosis affected more males (57.2%) and adults from 19-65 years (47.9%) and showed a bimodal seasonality with summer and winter peaks. In 11,251 routine stool cultures, Campylobacter spp. was detected in 4.6%, mostly C. jejuni (89.6%). Among 4533 samples tested by GMP and culture in parallel, GMP showed a superior sensitivity (99.1% versus 50%, respectively). The study suggests that Campylobacter spp. is the most frequent bacterial enteropathogen in Chile.

Evaluation of two fluorescence immunoassays for the rapid detection of SARS-CoV-2 antigen-new tool to detect infective COVID-19 patients.

BACKGROUND: Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) is currently the only recommended diagnostic method for SARS-CoV-2. However, rapid immunoassays for SARS-CoV-2 antigen could significantly reduce the COVID-19 burden currently weighing on laboratories around the world. METHODS: We evaluated the performance of two rapid fluorescence immunoassays (FIAs), SOFIA SARS Antigen FIA (Quidel Corporation, San Diego, CA, USA) and STANDARD F COVID-19 Ag FIA (SD Biosensor Inc., Gyeonggi-do, Republic of Korea), which use an automated reader. The study used 64 RT-PCR characterized clinical samples (32 positive; 32 negative), which consisted of nasopharyngeal swabs in universal transport medium. RESULTS: Of the 32 positive specimens, all from patients within 5 days of symptom onset, the Quidel and SD Biosensor assays detected 30 (93.8%) and 29 (90.6%) samples, respectively. Among the 27 samples with high viral loads (Ct ≤ 25), the two tests had a sensitivity of 100%. Specificity was 96.9% for both kits. CONCLUSION: The high performance of the evaluated FIAs indicates a potential use as rapid and PCR-independent tools for COVID-19 diagnosis in early stages of infection. The excellent sensitivity to detect cases with viral loads above ~106 copies/mL (Ct values ≤ 25), the estimated threshold of contagiousness, suggests that the assays might serve to rapidly identify infective individuals.

[Interactive, semi-automatized and open source computational model applied to respiratory viruses surveillance]. / Modelo computacional interactivo, semi-automatizado y de código abierto aplicado a la vigilancia de virus respiratorios.

Acute respiratory infections (ARI) are an important cause of morbidity and mortality worldwide, affecting mainly children and the elderly. They are associated with a high economic burden, increased number of medical visits and hospitalizations. The surveillance of the circulation of respiratory viruses can reduce the health care associated costs, and to optimize the health response. A platform based on R and its package Shiny was designed, to create an interactive and friendly web interface for gathering, analysis and publication of the data. The data from the Chilean metropolitan respiratory viruses surveillance network, available since 2006, was uploaded into the platform. Using this platform, the researcher spends less than 1 minute to upload the data, and the analysis and publication is immediate, available to be seen by any user with a device connected to Internet, who can choose the variables to be displayed. With a very low cost, in a short time, and using the R programming language, it was possible to create a simple, and interactive platform, considerably decreasing the upload and analysis time, and increasing the impact and availability of this surveillance.

Evaluation of a novel antigen-based rapid detection test for the diagnosis of SARS-CoV-2 in respiratory samples.

OBJECTIVES: In the context of the coronavirus disease 2019 (COVID-19) pandemic, the development and validation of rapid and easy-to-perform diagnostic methods are of high priority. This study was performed to evaluate a novel rapid antigen detection test (RDT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory samples. METHODS: The fluorescence immunochromatographic SARS-CoV-2 antigen test (Bioeasy Biotechnology Co., Shenzhen, China) was evaluated using universal transport medium with nasopharyngeal (NP) and oropharyngeal (OP) swabs from suspected COVID-19 cases. Diagnostic accuracy was determined in comparison to SARS-CoV-2 real-time (RT)-PCR. RESULTS: A total of 127 samples were included; 82 were RT-PCR-positive. The median patient age was 38 years, 53.5% were male, and 93.7% were from the first week after symptom onset. Overall sensitivity and specificity were 93.9% (95% confidence interval 86.5-97.4%) and 100% (95% confidence interval 92.1-100%), respectively, with a diagnostic accuracy of 96.1% and Kappa coefficient of 0.9. Sensitivity was significantly higher in samples with high viral loads. CONCLUSIONS: The RDT evaluated in this study showed a high sensitivity and specificity in samples mainly obtained during the first week of symptoms and with high viral loads, despite the use of a non-validated sample material. The assay has the potential to become an important tool for early diagnosis of SARS-CoV-2, particularly in situations with limited access to molecular methods.

Modelo computacional interactivo, semi-automatizado y de código abierto aplicado a la vigilancia de virus respiratorios / Interactive, semi-automatized and open source computational model applied to respiratory viruses surveillance

Resumen Las infecciones respiratorias agudas (IRA) causadas por virus son una importante causa de morbilidad y mortalidad en el mundo, afectando principalmente a niños y adultos mayores. Se asocian a un alto número de consultas y hospitalizaciones, a una significativa sobrecarga del sistema de salud y a un alto costo económico. La vigilancia de virus respiratorios tiene el potencial de ayudar a optimizar la respuesta sanitaria, garantizar la disponibilidad de recursos humanos, racionalizar los recursos y disminuir los costos asociados a la atención en salud. Con el objetivo de optimizar la recolección y visualización de los datos de nuestro actual sistema de vigilancia de virus respiratorios, se diseñó una plataforma basada en R y sus paquetes Shiny, que permite la creación de una interfase web interactiva y amigable para la recolección, análisis y publicación de los datos. Se ingresaron a esta plataforma los datos de la red de vigilancia metropolitana de virus respiratorios disponibles desde 2006. En esta plataforma, el investigador demora menos de un minuto en registrar los datos. El análisis y publicación es inmediato, llegando a cualquier usuario con un dispositivo conectado a Internet, quien puede elegir las variables a consultar. Con un costo muy bajo, en poco tiempo y utilizando el lenguaje de programación R, se logró crear un sistema simple e interactivo, disminuyendo el tiempo de carga y análisis de datos de forma considerable, posiblemente aumentando el impacto y la disponibilidad de esta vigilancia.

Abstract Acute respiratory infections (ARI) are an important cause of morbidity and mortality worldwide, affecting mainly children and the elderly. They are associated with a high economic burden, increased number of medical visits and hospitalizations. The surveillance of the circulation of respiratory viruses can reduce the health care associated costs, and to optimize the health response. A platform based on R and its package Shiny was designed, to create an interactive and friendly web interface for gathering, analysis and publication of the data. The data from the Chilean metropolitan respiratory viruses surveillance network, available since 2006, was uploaded into the platform. Using this platform, the researcher spends less than 1 minute to upload the data, and the analysis and publication is immediate, available to be seen by any user with a device connected to Internet, who can choose the variables to be displayed. With a very low cost, in a short time, and using the R programming language, it was possible to create a simple, and interactive platform, considerably decreasing the upload and analysis time, and increasing the impact and availability of this surveillance.

Genotipo y fenotipo de la enzima tiopurina metiltransferasa en población chilena / Phenotype and genotype of thiopurine methyltransferase in Chilean individuals

Background: Thiopurines (azathioprine and 6-mercaptopurine) are highly effective medications but with potential adverse effects. Thiopurine methyltransferase (TMPT) is the key enzyme in their pharmacokinetics and is genetically regulated. A low activity of TPMT is associated with myelotoxicity. The genotype and enzyme activity can vary by ethnicity. Aim: To study the activity and genotype of TPMT in a group of Chilean subjects. Material and Methods: In 200 healthy adult blood donors, TPMT activity was determined by high performance liquid chromatography (HPLC). Deficient, low, normal or high levels were defined when enzymatic activity was < 5, 6-24,25-55 and > 56 nmol/grHb/h, respectively. Genotyping of TPMT (*1, *2, *3A, *3B, *3C) was performed by PCR. Results: Seventy seven women (38.5%) and 123 men (61.5%), with an average age of 34.9 years were studied. Eighteen subjects (9%) had a low enzymatic activity, 178 (89%) had normal activity, 4 (2%) had high activity and no genotype deficient subjects were identified. The wild type genotype (*1) was found in 184 (92%) individuals and 16 (8%) were heterozygous for the variants: *2 (n = 2), *3A (n = 13) and *3C (n = 1). No homozygous subjects for these variants were identified. Wild type genotype had an increased enzymatic activity (40.8 ± 7.2 nmol/gHb/h) compared to heterozygous group (21.2 ± 3 nmol/ gHb/h; p < 0.001). Conclusions: Less than 10% of a Chilean population sample has a low enzymatic activity or allelic variants in the TPMT gene, supporting the use of thiopurines according to international recommendations.

[Phenotype and genotype of thiopurine methyltransferase in Chilean individuals]. / Genotipo y fenotipo de la enzima tiopurina metiltransferasa en población chilena.

BACKGROUND: Thiopurines (azathioprine and 6-mercaptopurine) are highly effective medications but with potential adverse effects. Thiopurine methyltransferase (TMPT) is the key enzyme in their pharmacokinetics and is genetically regulated. A low activity of TPMT is associated with myelotoxicity. The genotype and enzyme activity can vary by ethnicity. AIM: To study the activity and genotype of TPMT in a group of Chilean subjects. MATERIAL AND METHODS: In 200 healthy adult blood donors, TPMT activity was determined by high performance liquid chromatography (HPLC). Deficient, low, normal or high levels were defined when enzymatic activity was < 5, 6-24,25-55 and > 56 nmol/grHb/h, respectively. Genotyping of TPMT (*1, *2, *3A, *3B, *3C) was performed by PCR. RESULTS: Seventy seven women (38.5%) and 123 men (61.5%), with an average age of 34.9 years were studied. Eighteen subjects (9%) had a low enzymatic activity, 178 (89%) had normal activity, 4 (2%) had high activity and no genotype deficient subjects were identified. The wild type genotype (*1) was found in 184 (92%) individuals and 16 (8%) were heterozygous for the variants: *2 (n = 2), *3A (n = 13) and *3C (n = 1). No homozygous subjects for these variants were identified. Wild type genotype had an increased enzymatic activity (40.8 ± 7.2 nmol/gHb/h) compared to heterozygous group (21.2 ± 3 nmol/ gHb/h; p < 0.001). CONCLUSIONS: Less than 10% of a Chilean population sample has a low enzymatic activity or allelic variants in the TPMT gene, supporting the use of thiopurines according to international recommendations.

Plasminogen activator inhibitor type 1 serum levels and 4G/5G gene polymorphism in morbidly obese Hispanic patients with non-alcoholic fatty liver disease.

BACKGROUND: The plasminogen activator inhibitor type-1 (PAI-1) has been implicated in the regulation of fibrinolysis and extracellular matrix components. The single base pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene influences PAI-1 synthesis and may modulate hepatic fibrogenesis. AIM: To evaluate the influence of PAI-1 serum levels and 4G/5G polymorphism on the risk of liver fibrosis associated to non-alcoholic fatty liver disease (NAFLD) in morbidly obese patients. MATERIAL AND METHODS: Case-control study of 50 obese patients undergoing bariatric surgery and 71 non-obese subjects matched by age and sex. Anthropometric and biochemical measurements were performed, including PAI-1 serum levels. Genomic DNA was obtained to assess the presence of 4G/5G polymorphism. RESULTS: BMI, insulinemia, triglycerides, HOMA-IR, hypertension and diabetes were significantly higher in obese patients compared to control subjects. PAI-1 serum levels observed in obese patients were significantly lower (10.63 ± 4.82) compared to controls (14.26 ± 11.4; p < 0.05). No differences were observed in the PAI-1 4G/5G promoter genotypes frequencies (p = 0.12). No differences were observed in PAI-1 plasma levels among obese patients with liver fibrosis (10.64 ± 4.35) compared to patients without liver fibrosis (10.61 ± 5.2; p = 0.985). PAI-1 4G/5G promoter genotypes frequencies were similar in patients with or without liver fibrosis associated to NASH (p = 0.6). CONCLUSIONS: Morbidly obese patients had significantly lower PAI-1 serum levels with similar PAI-1 4G/5G genotypes frequencies compared to non-obese subjects. The frequency of 4G/5G genotypes in Chilean Hispanic healthy subjects was similar to that described in other populations. No association was found between PAI-1 serum levels or 4G/5G genotype with liver fibrosis in obese patients.

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation.

The HCV internal ribosome entry site (IRES) spans a region of approximately 340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg(2+) ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.

Influence of extrahepatic viral infection on the natural history of hepatitis C.

HCV is primarily hepatotropic, but there is mounting evidence pointing to infection and replication of extrahepatic sites. Here we evaluated the occurrence of HCV infection of peripheral blood mononuclear cells (PBMC) and explored the possible association between viral extrahepatic infection and the natural history of the disease. Forty seven Chilean, HCV infected, treatment naïve patients were included in the study. HCV RNA was isolated from plasma and PBMC and subsequently reverse transcribed, amplified and sequenced. Most patients harbored HCV 1b genotype and the most common route of infection showed to be blood transfusion. HCV RNA was readily detected in PBMCs of 34 out of the 47 patients (72%). We report that HCV sequences found in PBMC differ from those in plasma of the same subjects strongly suggesting HCV compartmentalization. In addition, we found that patients with detectable HCV RNA in PBMC had a tendency for being more likely cirrhotic [OR 3.8 (95% CI: 0.98 to 14)]. In conclusion, this study provides further arguments for the existence of HCV infection of extrahepatic sites and suggests that extrahepatic infection could be a factor influencing the natural history of the disease.

Bucillamine induces glutathione biosynthesis via activation of the transcription factor Nrf2.

The properties of bucillamine, a synthetic antioxidant, have been attributed mainly to the donation of thiol groups to glutathione (GSH). We recently demonstrated that glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme of GSH biosynthesis, and the multidrug-resistance-associated protein 2 (Mrp2/MRP2) are coordinately induced in response to xenobiotic through the activation of the antioxidant-response element (ARE) by nuclear factor-erythroid 2 p45-related factor (Nrf2). We tested the hypothesis that bucillamine and its oxidized metabolite SA 981 also activate the Nrf2 pathway, thereby increasing glutathione biosynthesis in human HepG2 and murine Hepa 1-6 hepatoma cell lines, through the induction of the GCLC enzyme as well as the Mrp2/MRP2 transporter, which mediates the excretion of glutathione and its conjugates from hepatocytes. Both bucillamine and SA 981 produced a significant dose-dependent increase in the mRNA levels of Mrp2/MRP2 and GCLC after 24 h. The levels of the transcription factor Nrf2 in the nuclei were maximal at 3 h, remained elevated at 6 h, and decreased to control values at 24 h in both cell lines. Moreover, both bucillamine and SA 981 significantly increased the expressions of Mrp2/MRP2 and GCLC proteins in both cell lines. Finally, in both cell lines, bucillamine and SA 981 increased the GSH content two- to three-fold. These results demonstrate that bucillamine and SA 981 activate the ARE-ARE pathway increasing the expression of ARE-driven genes such as those of GCLC and Mrp2/MRP2. The role of bucillamine as a chemopreventive agent against cancer remains to be elucidated.

C(-106)T polymorphism of the aldose reductase gene and the progression rate of diabetic retinopathy.

PURPOSE: To study the C(-106)T polymorphism in the promoter of the aldose reductase (ALR2) gene: (a) its local prevalence and (b) its modulation of the susceptibility for developing retinopathy. METHODS: DNAs of 96 control subjects and 53 long-standing (duration 17.9+/-5.4 years) type-2 diabetic patients were analyzed by PCR-RFLP with BfaI enzyme. Retinopathy was graded with 2-eye, 7-field fundus color photography. The IMF-HbA1c was the arithmetic mean of all HbA1c's of each patient. RESULTS: The genotypes in the controls were CC=57 (59.4%), CT=32 (33.3%) and TT=7 (7.3%), with Hardy-Weinberg chi(2)=0.793 (p>0.50). Among 53 diabetics, CC=24 (45.3%), CT=26 (49.0%) and TT=3 (5.7%). The correlation between IMF-HbA1c and retinopathy progression rate was significant on CC (r=0.6102, p=0.0072) but not in CT+TT genotypes (r=0.26, p=0.1811). CONCLUSIONS: In Chilean adults, the frequency of the C(-106)T polymorphism of the ALR2 gene was similar to that reported by others. Type-2 diabetics with the CC genotype were more susceptible for developing retinopathy as a result of chronic hyperglycemia than those with the CT or TT genotype.

Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene.

The Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) transcription factor regulates gene expression of the GCLC (glutamate-cysteine ligase catalytic subunit), which is a key enzyme in glutathione synthesis, and GSTs (glutathione S-transferases) via the ARE (antioxidant-response element). The Mrp2 (multidrug-resistance protein 2) pump mediates the excretion of GSH and GSSG excretion as well as endo- and xeno-biotics that are conjugated with GSH, glucuronate or sulphate. Considering that Mrp2 acts synergistically with these enzymes, we hypothesized that the regulation of Mrp2 gene expression is also dependent on Nrf2. Using BHA (butylated hydroxyanisole), which is a classical activator of the ARE-Nrf2 pathway, we observed an increase in the transcriptional activity of Mrp2, GCLC and Gsta1/Gsta2 genes in the mouse liver. A similar pattern of co-induction of Mrp2 and GCLC genes was also observed in mouse (Hepa 1-6) and human (HepG2) hepatoma cells treated with BHA, beta-NF (beta-naphthoflavone), 2,4,5-T (trichlorophenoxyacetic acid) or 2AAF (2-acetylaminofluorene), suggesting that these genes share common mechanism(s) of transcriptional activation in response to exposure to xenobiotics. To define the mechanism of Mrp2 gene induction, the 5'-flanking region of the mouse Mrp2 gene (2.0 kb) was isolated, and two ARE-like sequences were found: ARE-2 (-1391 to -1381) and ARE-1 (-95 to -85). Deletion analyses demonstrated that the proximal region (-185 to +99) contains the elements for the basal expression and xenobiotic-mediated induction of the Mrp2 gene. Gel-shift and supershift assays indicated that Nrf2-protein complexes bind ARE sequences of the Mrp2 promoter, preferentially to the ARE-1 sequence. Overexpression of Nrf2 increased ARE-1-mediated CAT (chloramphenicol acetyltransferase) gene activity, while overexpression of mutant Nrf2 protein repressed the activity. Thus Nrf2 appears to regulate Mrp2 gene expression via an ARE element located at the proximal region of its promoter in response to exposure to xenobiotics.

[Polymorphisms of the multiple drug resistance gene (MDR1) in Mapuche, Mestizo and Maori populations in Chile]. / Polimorfismos del gen de resistencia a múltiples drogas (MDR1) en poblaciones chilenas: mapuche, mestiza y maorí.

BACKGROUND: There are significant differences in drug responses among different ethnic groups. The multidrug transporter P-gp, encoded by the MDR1 gene, plays a key role in determining drug bioavailability, and an association between a polymorphism in exon 26 (C3435T) and lower P-gp expression has been found. The co-segregation of this polymorphism with the polymorphism in exon 12 (C1236T) and in exon 21 (G2677T/A) determines several MDR1 haplotypes in humans. AIM: To characterize the polymorphisms of exons 26, 21 and 12 of the MDR1 gene in different Chilean populations. MATERIAL AND METHODS: Using a polymerase chain reaction and restriction fragment length polymorphism technique, we studied the allelic frequencies and the distribution of MDR1 haplotypes in 3 Chilean populations: Mestizo (n=104), Mapuche (n=96, living in the National Reservation of the Huapi Island, Ranico Lake) and Maori (n=52, living in Eastern Island). RESULTS: The frequency of the normal MDR1*1 haplotype, without mutations, was lower in Mapuches than in Mestizos or Maoris (p<0.005) but similar to that reported in Asian population (p=0.739), probably due to the Asian origin of the Amerindian populations. In addition, the MDR1*l haplotype fequency hin Mestizos was similar to the frequency reported in Caucasians (p=0.49), in agreement with the origin of our population, with a strong influence of Caucasian genes from the Spanish conquerors. The MDR1*2 haplotype distribution, with the three polymoyphisms and probably lower multidrug transporter expression, was similar in the three Chilean populations studied (p>0.0.5), but lower than the frequencies reported in Caucasians or Asians (p<0.05). CONCLUSIONS: We found significant differences in the frequencies of genetic polymorphisms of the MDR1 gene in Chilean populations, related to the ethnic origins of our ancestors.