A precision overview of genomic resistance screening in Ecuadorian isolates of Mycobacterium tuberculosis using web-based bioinformatics tools.

INTRODUCTION: Tuberculosis (TB) is among the deadliest diseases worldwide, and its impact is mainly due to the continuous emergence of resistant isolates during treatment due to the laborious process of resistance diagnosis, nonadherence to treatment and circulation of previously resistant isolates of Mycobacterium tuberculosis. In this study, we evaluated the performance and functionalities of web-based tools, including Mykrobe, TB-profiler, PhyResSE, KvarQ, and SAM-TB, for detecting resistance in 88 Ecuadorian isolates of Mycobacterium tuberculosis drug susceptibility tested previously. Statistical analysis was used to determine the correlation between genomic and phenotypic analysis. Our results showed that with the exception of KvarQ, all tools had the highest correlation with the conventional drug susceptibility test (DST) for global resistance detection (98% agreement and 0.941 Cohen's kappa), while SAM-TB, PhyResSE, TB-profiler and Mykrobe had better correlations with DST for first-line drug analysis individually. We also identified that in our study, only 50% of mutations characterized by the web-based tools in the rpoB, katG, embB, pncA, gyrA and rrs regions were canonical and included in the World Health Organization (WHO) catalogue. Our findings suggest that SAM-TB, PhyResSE, TB-profiler and Mykrobe were efficient in determining canonical resistance-related mutations, but more analysis is needed to improve second-line detection. Improving surveillance programs using whole-genome sequencing tools for first-line drugs, MDR-TB and XDR-TB is essential to understand the molecular epidemiology of TB in Ecuador. IMPORTANCE: Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, most commonly affects the lungs and is often spread through the air when infected people cough, sneeze, or spit. However, despite the existence of effective drug treatment, patient adherence, long duration of treatment, and late diagnosis have reduced the effectiveness of therapy and increased drug resistance. The increase in resistant cases, added to the impact of the COVID-19 pandemic, has highlighted the importance of implementing efficient and timely diagnostic methodologies worldwide. The significance of our research is in evaluating and identifying a more efficient and user-friendly web-based tool to characterize resistance in Mycobacterium tuberculosis by whole-genome sequencing, which will allow more routine application to improve TB strain surveillance programs locally.

Comparative genomics of drug-resistant strains of Mycobacterium tuberculosis in Ecuador.

BACKGROUND: Tuberculosis is a serious infectious disease affecting millions of people. In spite of efforts to reduce the disease, increasing antibiotic resistance has contributed to persist in the top 10 causes of death worldwide. In fact, the increased cases of multi (MDR) and extreme drug resistance (XDR) worldwide remains the main challenge for tuberculosis control. Whole genome sequencing is a powerful tool for predicting drug resistance-related variants, studying lineages, tracking transmission, and defining outbreaks. This study presents the identification and characterization of resistant clinical isolates of Mycobacterium tuberculosis including a phylogenetic and molecular resistance profile study by sequencing the complete genome of 24 strains from different provinces of Ecuador. RESULTS: Genomic sequencing was used to identify the variants causing resistance. A total of 15/21 isolates were identified as MDR, 4/21 as pre-XDR and 2/21 as XDR, with three isolates discarded due to low quality; the main sub-lineage was LAM (61.9%) and Haarlem (19%) but clades X, T and S were identified. Of the six pre-XDR and XDR strains, it is noteworthy that five come from females; four come from the LAM sub-lineage and two correspond to the X-class sub-lineage. A core genome of 3,750 genes, distributed in 295 subsystems, was determined. Among these, 64 proteins related to virulence and implicated in the pathogenicity of M. tuberculosis and 66 possible pharmacological targets stand out. Most variants result in nonsynonymous amino acid changes and the most frequent genotypes were identified as conferring resistance to rifampicin, isoniazid, ethambutol, para-aminosalicylic acid and streptomycin. However, an increase in the resistance to fluoroquinolones was detected. CONCLUSION: This work shows for the first time the variability of circulating resistant strains between men and women in Ecuador, highlighting the usefulness of genomic sequencing for the identification of emerging resistance. In this regard, we found an increase in fluoroquinolone resistance. Further sampling effort is needed to determine the total variability and associations with the metadata obtained to generate better health policies.

Omicron Sub-Lineages (BA.1.1.529 + BA.*) Current Status in Ecuador.

The Omicron variant of SARS-CoV-2 is the latest pandemic lineage causing COVID-19. Despite having a vaccination rate ≥85%, Ecuador recorded a high incidence of Omicron from December 2021 to March 2022. Since Omicron emerged, it has evolved into multiple sub-lineages with distinct prevalence in different regions. In this work, we use all Omicron sequences from Ecuador available at GISAID until March 2022 and the software Nextclade and Pangolin to identify which lineages circulate in this country. We detected 12 different sub-lineages (BA.1, BA.1.1, BA.1.1.1, BA.1.1.14, BA.1.1.2, BA.1.14, BA.1.15, BA.1.16, BA.1.17, BA.1.6, BA.2, BA.2.3), which have been reported in Africa, America, Europe, and Asia, suggesting multiple introduction events. Sub-lineages BA.1 and BA.1.1 were the most prevalent. Genomic surveillance must continue to evaluate the dynamics of current sub-lineages, the early introduction of new ones and vaccine efficacy against evolving SARS-CoV-2.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic diversity through space and time can reveal trends in virus importation and domestic circulation and permit the exploration of questions regarding the early transmission dynamics. Here, we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the coronavirus-19 pandemic. We generated and analysed 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylogeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions, with differential degrees of persistence and national dissemination.

SARS-CoV-2 genome sequencing from COVID-19 in Ecuadorian patients: a whole country analysis.

SARS-CoV-2, the etiological agent of COVID-19, was first described in Wuhan, China in December 2019 and has now spread globally. Ecuador was the second country in South America to confirm cases and Guayaquil was one of the first cities in the world to experience high mortality due to COVID-19. The aim of this study was to describe the lineages circulating throughout the country and to compare the mutations in local variants, to the reference strain. In this work we used the MinION platform (Oxford Nanopore Technologies) to sequence the whole SARS-CoV-2 genomes of 119 patients from all provinces of Ecuador, using the ARTIC network protocols. Our data from lineage assignment of the one hundred and nineteen whole genomes revealed twenty different lineages. All genomes presented differences in the S gene compared to the Wuhan reference strain, being the D614G amino acid replacement the most common change. The B.1.1.119 lineage was the most frequent and was found in several locations in the Coast and Andean region. Three sequences were assigned to the new B.1.1.7 lineage. Our work is an important contribution to the understanding of the epidemiology of SARS-CoV-2 in Ecuador and South America.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of SARS-CoV-2 genetic diversity through space and time can reveal trends in virus importation and domestic circulation, and permit the exploration of questions regarding the early transmission dynamics. Here we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the COVID-19 pandemic. We generate and analyse 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylgeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions (NPIs), with differential degrees of persistence and national dissemination.

Description of the semen quality from men treated in an assisted reproduction center in Guayaquil, Ecuador. / Caracterización de la calidad del semen en hombres atendidos en un centro de reproducción asistida en Guayaquil, Ecuador.

In order to characterize the quality of semen from men in an assisted reproduction center in the city of Guayaquil (Ecuador), 204 semen samples were collected from patients with fertility disorders aged 20 to 57 years, who were admitted between May 2017 and September 2018. A basic spermogram was performed on each sample, following the fabricant recommendations for the examination and processing of human semen. It was found that 27.4% of the samples presented normozoospermia. Among the disorders, it was found that 27.9% had teratozoospermia, 8.8% had oligoteratozoospermia and a higher number of patients were found to be between 30 and 39 years old. A high percentage of patients presented sperm morphology and quality values below the reference limits established by the World Health Organization.

Con el objetivo de caracterizar la calidad seminal de hombres en un centro de reproducción asistida de la ciudad Guayaquil (Ecuador), se colectaron 204 muestras de semen de pacientes con problemas de fertilidad de entre 20 y 57 años, atendidos entre mayo de 2017 y septiembre de 2018. Se realizó un espermograma básico a cada muestra, siguiendo las recomendaciones del manual para la examinación y procesamiento de semen humano. El 27,4% de las muestras presentó normozoospermia. Dentro de las alteraciones la teratozoospermia fue de 27,9%, oligoteratozoospermia del 8,8%, evidenciándose mayor número en pacientes de 30 a 39 años. Un alto porcentaje de pacientes presentan una calidad del semen y morfología espermática por debajo los limites de referencia establecidos por la Organización Mundial de la Salud.

Caracterización de la calidad del semen en hombres atendidos en un centro de reproducción asistida en Guayaquil, Ecuador / Description of the semen quality from men treated in an assisted reproduction center in Guayaquil, Ecuador

RESUMEN Con el objetivo de caracterizar la calidad seminal de hombres en un centro de reproducción asistida de la ciudad Guayaquil (Ecuador), se colectaron 204 muestras de semen de pacientes con problemas de fertilidad de entre 20 y 57 años, atendidos entre mayo de 2017 y septiembre de 2018. Se realizó un espermograma básico a cada muestra, siguiendo las recomendaciones del manual para la examinación y procesamiento de semen humano. El 27,4% de las muestras presentó normozoospermia. Dentro de las alteraciones la teratozoospermia fue de 27,9%, oligoteratozoospermia del 8,8%, evidenciándose mayor número en pacientes de 30 a 39 años. Un alto porcentaje de pacientes presentan una calidad del semen y morfología espermática por debajo los limites de referencia establecidos por la Organización Mundial de la Salud.

ABSTRACT In order to characterize the quality of semen from men in an assisted reproduction center in the city of Guayaquil (Ecuador), 204 semen samples were collected from patients with fertility disorders aged 20 to 57 years, who were admitted between May 2017 and September 2018. A basic spermogram was performed on each sample, following the fabricant recommendations for the examination and processing of human semen. It was found that 27.4% of the samples presented normozoospermia. Among the disorders, it was found that 27.9% had teratozoospermia, 8.8% had oligoteratozoospermia and a higher number of patients were found to be between 30 and 39 years old. A high percentage of patients presented sperm morphology and quality values below the reference limits established by the World Health Organization.

Análisis genético de la resistencia a isoniacida en cepas de Mycobacterium tuberculosis / Genetic analysis of isoniazid resistance in strains of Mycobacterium tuberculosis

Objetivo: Analizar genéticamente la resistencia a Isoniacida en cepas de Mycobacterium tuberculosis mediante PCR-RFLP de la región S315T del gen katG. Materiales y métodos: El estudio se realizó a partir de cultivos positivos de Mycobacterium tuberculosis receptados en el Centro de Referencia Nacional de Micobacterias, durante el período 2013 ­ 2014. El ADN extraído fue cuantificado y evaluada su pureza, por espectrofotometría. Para determinar el polimorfismo en la región 315 del gen katG a partir de un producto de amplificación de 630 pb se realizó digestiones con las enzimas de restricción MspI y SatI. Resultados: Del total de 498 cepas analizadas, 215 cepas presentaron características fenotípicas de resistencia a isoniacida (32,6 % monorresistencia, 19,5 % MDR y 47,9 % polirresistencia), 283 cepas eran sensibles. 251 cepas correspondieron a pacientes vírgenes al tratamiento (VT); 174 fueron pacientes antes tratados (AT) y 73 fueron pacientes se encontraban con tratamiento (CT). La mayoría de los casos provenía de la provincia del Guayas (77,2 %). La PCR-RFLP-SatI presentó alto porcentaje de sensibilidad (98,6 %) y especificidad (98,2 %), mientras que con la enzima MspI el porcentaje de sensibilidad fue 88,8 % y 7,4 % de especificidad. Conclusión: La PCR-RFLP-SatI demostró ser específica y económica para la detección de resistencia a isoniacida, proporcionando resultados de forma rápida, la aplicación de esta técnica como apoyo para el diagnóstico permitiría al paciente acceder a un tratamiento más oportuno.

Objective: Genetically analyze the Isoniazid resistance in Mycobacterium tuberculosis cultures by PCR-RFLP of the S315T region of the atG. Materials and methods: The study was carried out in positive cultures of Mycobacterium tuberculosis, received at the National Reference Center of Mycobacteria, during the period 2013-2014. The DNA extracted was quantified and its purity was evaluated by spectrophotometry. To determine the polymorphism in the 315 region of the at G gene, digestions were made with the restriction enzymes MspI and SatI from a 630 bp amplification product. Results: Of 498 culture strains analyzed, 215 strains showed phenotypic characteristics of resistance to Isoniazid (32,6 % monoresistance, 19,5 % MDR and 47,9 % polyresistance) and 283 strains were sensitive. 251 strains corresponded to virgin patients to treatment (VT); 174 were patients before treated (AT) and 73 were patients treated (CT). The majority of cases came from the province of Guayas (77,2 %). The PCR-RFLP- SatI presented a high percentage of sensitivity (98,6 %) and specificity (98,2 %), while with the MspI enzyme the sensitivity percentage was 88,8 % and 7,4 % specificity. Conclusion: The PCR-RFLP SatI proved to be specific and economical for the detection of resistance to isoniazid, providing results quickly, the application of this technique as a support for the diagnosis would allow the patient to access a more timely treatment.

Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP / Determination of S315T mutation within the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolate by PCR-RFLP

Objetivo: determinar la mutación S315T del gen katG en aislados de Mycobacterium tuberculosis resistentes a isoniacida mediante la técnica PCR-RFLP. Materiales y métodos: A partir de 68 aislados de Mycobacterium tuberculosis se realizó el análisis de polimorfismo en productos de amplificación de 1054 y 630 pb que contenían la mutación S315T del gen katG mediante PCR-RFLP empleando las enzimas de restricción MspI y SatI. Mediante SPSS se determinó sensibilidad, especificidad, valores predictivo positivo y negativo, coeficientes de probabilidad positivo y negativo. Resultados: El 74,46% de aislados fenotípicamente resistentes y 4,76% fenotípicamente sensibles presentaron la mutación del gen katG S315T. La PCR-RFLP para S315T del gen katG presentó 85,4% de sensibilidad y 95,2% de especificidad con MspI y 85,4% de sensibilidad y 94,4% de especificidad con SatI. Discusión: La PCR-RFLP tiene una alta capacidad resolutiva que depende de la enzima que se emplee como se observó en estudios previos. La presencia de la mutación S315T en pacientes vírgenes al tratamiento sugiere la circulación de aislados resistentes a Isoniacida. Conclusión: La PCR-RFLP resultó una alternativa válida y rápida para el diagnóstico de la resistencia a isoniacida, mediante la detección de la mutación S315T del gen katG en comparación con el método convencional de las proporciones.

Objetive: To determine the S315T mutation of the katG gene in Mycobacterium tuberculosis isoniazid-resistant isolates by PCR-RFLP. Materials and Methods: Polymorphism analysis of 1054 and 630 bp products containing the S315T mutation of the katG gene was performed by PCR-RFLP using the MspI and SatI restriction enzymes from 68 Mycobacterium tuberculosis isolates. Sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio were determined using SPSS. Results: 74.46% of isoniazid-resistant and 4.76% of isoniazid-sensitive isolates showed the S315T mutation in katG gene. The PCR-RFLP for S315T of the katG gene had 85.4% sensitivity and 95.2% specificity with MspI and 85.4% sensitivity and 94.4% specificity with SatI. Discussion: The PCR-RFLP has a high resolutive capacity that depends on the enzyme that is used as it was observed in previous studies. The presence of the S315T mutation in treatment-naive patients suggests the circulation of isolates resistant to isoniazid. Conclusion: PCR-RFLP is a valid and rapid alternative for the diagnosis of isoniazid resistance, by detection of S315T mutation in the katG gene compared to the conventional method of proportions.

Utilidad de la Proteína Epididimal Humana 4 (HE4) en la detección de Cáncer de ovario / Usefulness of the Human Epididymis protein 4 (HE4) in the detection of ovarian cancer

Introducción: El cáncer de ovario epitelial aunque tiene baja prevalencia está considerado entre las malignidades ginecológicas más letales por su alta mortalidad. El interés en la detección temprana del cáncer de ovario como mecanismo para lograr la reducción de la mortalidad ha crecido con el descubrimiento de biomarcadores tumorales séricos asociados a tumores malignos. El presente estudio plantea determinar la eficacia del uso del biomarcador HE4 para la detección precoz de cáncer epitelial de ovario en estadios tempranos. Métodos: Se evaluaron pacientes con masas pélvicas entre abril de 2015 y marzo de 2016. Valores de sensibilidad, especificidad, predictivo positivo y negativo, razón de probabilidad positiva y negativa, y pruebas estadísticas fueron calculados para determinar la relación entre los estados menopáusicos, y los grupos de acuerdo con el resultado histológico (benigno, maligno y control) de HE4, CA125 y ROMA. Resultados: Ingresaron al estudio 53 pacientes. La proteína epididimal humana 4 - HE4 presentó un valor medio diferenciable que permite distinguir masas pélvicas malignas (HE4:7,19 (maligno) vs. 5,71 (benigno)), igualmente la combinación HE4 + ROMA presentan mayor sensibilidad y especificidad (S: 100 %; E: 94.29 %) que las combinaciones CA125 + HE4 y CA125 + ROMA (S: 80 % y 88.89 %; E: 75.76 % y 77.14 %). Conclusión: Los resultados sugieren que HE4 serviría como un biomarcador eficiente para la diferenciación de masas pélvicas en estadios tempranos y si se adiciona el estatus menopaúsico, e índice ROMA afianzaría los resultados, permitiendo la diferenciación del cáncer de ovario epitelial en estadios tempranos en el país.

Introduction: Although epithelial ovarian cancer (EOC) has a low prevalence, it is considered among the most lethal gynecological malignancies due to its high mortality. The interest in the early detection of ovarian cancer as a mechanism to achieve the reduction of mortality has grown with the discovery of serum tumor biomarkers associated with malignant tumors. The present study proposes to determine the efficacy of the use of the HE4 biomarker for the early detection of ovarian epithelial cancer in early stages. Methods: We evaluated 53 patients with pelvic masses between April 2015 and March 2016. Sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio, and statistical tests were calculated to determine the relationship between menopausal states, and groups according to the histological result (benign, malignant and control) of HE4, CA125 and ROMA. Results: The human epididymal protein 4 - HE4 presented a differentiable mean value that allows to distinguish malignant pelvic masses (HE4: 7.19 (malignant) vs. 5.71 (benign)), likewise the combination HE4 + ROMA present greater sensitivity and specificity (S: 100%; E: 94.29 %) than combinations CA125 + HE4 and CA125 + ROMA (S: 80% and 88.89 %; E: 75.76 % and 77.14 %). Conclusion: The results suggest that HE4 would serve as an efficient biomarker for the differentiation of pelvic masses in early stages and if menopausal status is added, and ROMA index would strengthen the results, allowing the differentiation of epithelial ovarian cancer in early stages in the country.