Brucellosis: an emerging disease in Portugal

Human brucellosis is one of the most common zoonosis worldwide. In Portugal, brucellosis is a notifiable disease in humans and the casuistic puts it among the three zoonosis with the highest incidence. Despite this, studies on prevalence of brucellosis in Portugal are scarce. The present study intends to evaluate the epidemiological situation of human brucellosis in Portugal and to identify the species associated with human cases. It also intend to investigate the origin of infection in humans using molecular typing studies and whole genome sequencing approaches. In this work, we propose study the genetic polymorphism of several virulence factors in Brucella spp. Our results showed that Brucella melitensis is the main species associated to human brucellosis and that there is a strong epidemiological link between many cases studies, whose originated small clusters and may even correspond to small outbreaks. In this study, it was possible to verify a strong phylogenetic proximity between isolated strains in the Mediterranean area (Spain, Greece and Italy) probably due to geographical, cultural and type of food proximity. In this work, using an advanced approach, new generation sequencing methodologies, we were able to validate the use of MLVA - 16, the gold standard for typing Brucella spp., using in silico extraction. Globally, the findings presented in this PhD thesis contribute for better understanding of the brucellosis situation in Portugal. The results may contribute to the implementation of a new laboratory tool to improve the epidemiological surveillance of brucellosis. Furthermore, is providing more accurate and quick information to the decision makers with responsibilities in the area of the implementation of measures of prevention and control of this disease in our Country, both in human and veterinary health, in line with the One Health approach

Detecting Biothreat Agents: the Portuguese National Reference Laboratory Response

At present, the threat of a biological terrorist event with a dangerous pathogen and its insidious impact are among the most, yet least understood, threats to civil society today. The use of manufactured weapon, such as bomb, will produce consequences limited in time hence the most of the damage occurs immediately. By contrast, the use of a biological weapon is an extended process whose scope and timing cannot be precisely controlled. Many biological agents could be used as biological weapons. The CDC bioterrorism list includes 45 biological agents that have been targeted as most likely to be used in bioterrorism attack. Of these, six have been designated as ‘‘high priority’’ agents: Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella spp., botulinum toxin produced by Clostridium botulinum and Variola major virus. Beside the pathogenic impact, the use of these agents will likely produce widespread panic, which will quickly overwhelm local law enforcement, as well as providers of health care. Since the majority of these agents require an incubation period before disease becomes noticed, the terrorists often escape without notice. The Portuguese National Institute of Health (PNIH) is prepared to recognize and respond to a biological event, including bioterrorism, having responsibilities in the determination of the etiological agent’s and its susceptibility to antibiotics. In order to ensure quick and reliable results, a laboratory algorithm was developed, taking in account the human and technical resources available. This algorithm was tested within a framework of an external quality control scheme and the results obtained demonstrated that the performance of our lab is at the same level of other European reference labs, even if the human, technical and financial resources are quite different. The PNIH also have the responsibility to inform other health and criminal authorities whenever an unusual biological agent is detected. Therefore, the role of PNIH is essential, promoting a rapid laboratory response and contributing to risk communication between the several authorities with responsibilities in the establishment of preparedness plans and measures that will contribute to control and mitigate the effects of bioterrorism attack on public health

Ebolavirus outbreak in West Africa: Portuguese laboratory response overview

The Ebola outbreak in West Africa was the largest and most complex outbreak since the virus was discovered in 1976. First cases were notified in March of 2014 and currently cases are still being reported in the affected countries. To respond to the epidemic of Ebola virus, Portugal created an coordination committee where the National Institute of Health, through the Emergency Response and Biopreparedness Unit (UREB), participated integrating the “Platform Response to Ebola Virus Disease”. This unit is the national reference laboratory for biological events or catastrophes and has skilled professionals, know-how, BSL-3 facilities, capacity to work 24h/7d and trained human resources to increase lab capacity in emergency situations. The laboratory diagnosis capacity includes the detection of bacteria, virus and toxins, which are considered bioterrorism agents, using Microbiology, Immunology and Molecular Biology techniques. elopment of national guidelines and establish an agreement with European reference BSL-4 laboratories for additional tests.N/

Demographic and Clinical Characteristics of Mpox Patients Attending an STD Clinic in Lisbon

(This article belongs to the Special Issue Human Monkeypox—an Emerging Zoonotic Disease and a Global Threat)Mpox is a viral disease caused by the monkeypox virus, which marked the year of 2022 with a global outbreak. While previously considered to be a zoonosis of almost exclusive animal-to-human transmission, the current outbreak has been attributed to human-to-human transmission, particularly sexual transmission. As a new sexually transmissible disease, we studied the epidemiological and clinical features, as well as the concomitant occurrence of other sexually transmissible diseases, treatment approach, and outcome of our 291 patients, in the current outbreak. We found a total of 169 concomitant sexually transmissible infections of bacterial and viral origins, corresponding to 107 patients. Neisseria gonorrhoeae was the most common agent, particularly in the anal location. With this work, we emphasize the need for a thorough epidemiological and medical history, as well as a concomitant complete laboratorial screening for other STIs in patients with confirmed or suspected mpox.info:eu-repo/semantics/publishedVersio

Whole Genome Sequencing-Based Approach for the Determination of Multiple Locus Variable Number Tandem Repeat Profiles

Funding Information: Funding: This work is a result of the GenomePT project (POCI-01- 0145-FEDER-022184), supported by the COMPETE 2020 – Operational Program for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Program (Lisboa2020), Algarve Portugal Regional Operational Program (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by the Fundação para a Ciência e a Tecnologia (FCT). The studies have arisen from the Project QUANDHIP (Chafea Grant Agreement no. 2010 21 02), which has been funded by the European Commission in the framework of the Health Program.Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding “boundaries” for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis.publishersversionpublishe

Burkholderia pseudomallei: primeiro caso de melioidose em Portugal

Objetivo: Este estudo apresenta o primeiro caso de melioidose em Portugal, revelando o importante papel da metodologia de Sequenciação Total do Genoma para a correta identificação e caracterização da estirpe isolada

Comparative complete scheme and booster effectiveness of COVID‐19 vaccines in preventing SARS‐CoV‐2 infections with SARS‐CoV‐2 Omicron (BA.1) and Delta (B.1.617.2) variants: A case–case study based on electronic health records

Background: Information on vaccine effectiveness in a context of novel variants of concern (VOC) emergence is of key importance to inform public health policies. This study aimed to estimate a measure of comparative vaccine effectiveness between Omicron (BA.1) and Delta (B.1.617.2 and sub-lineages) VOC according to vaccination exposure (primary or booster). Methods: We developed a case-case study using data on RT-PCR SARS-CoV-2-positive cases notified in Portugal during Weeks 49-51, 2021. To obtain measure of comparative vaccine effectiveness, we compared the odds of vaccination in Omicron cases versus Delta using logistic regression adjusted for age group, sex, region, week of diagnosis, and laboratory of origin. Results: Higher odds of vaccination were observed in cases infected by Omicron VOC compared with Delta VOC cases for both complete primary vaccination (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.8 to 2.4) and booster dose (OR = 5.2; 95% CI: 3.1 to 8.8), equivalent to reduction of vaccine effectiveness from 44.7% and 92.8%, observed against infection with Delta, to -6.0% (95% CI: 29.2% to 12.7%) and 62.7% (95% CI: 35.7% to 77.9%), observed against infection with Omicron, for complete primary vaccination and booster dose, respectively. Conclusion: Consistent reduction in vaccine-induced protection against infection with Omicron was observed. Complete primary vaccination may not be protective against SARS-CoV-2 infection in regions where Omicron variant is dominant.The acquisition of sequencing equipment and reagents used in this study by the Instituto Nacional de Saúde Doutor Ricardo Jorge was partially funded by the HERA project (grant no. 2021/PHF/23776), supported by the European Commission through the European Centre for Disease Control, and also partially funded by the Genome PT project (grant no. POCI-01-0145-FEDER-022184), supported by COMPETE 2020–Operational Programme for Competitiveness and Internationalisation, Lisboa Portugal Regional Operational Programme, Algarve Portugal Regional Operational, under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund, and by the Portuguese Science and Technology Foundation. The Algarve Biomedical Center Laboratory received public funding through the Project ALG-D2-2021-06 Variants Screen in Southern Portugal–Monitoring Variants of Concern in Southern Portugal and the Portuguese Science and Technology Foundation national support through the Comprehensive Health Research Center (grant no. UIDP/04923/2020).info:eu-repo/semantics/publishedVersio

Comparative complete scheme and booster effectiveness of COVID‐19 vaccines in preventing SARS‐CoV‐2 infections with SARS‐CoV‐2 Omicron (BA.1) and Delta (B.1.617.2) variants: A case–case study based on electronic health records

Background: Information on vaccine effectiveness in a context of novel variants of concern (VOC) emergence is of key importance to inform public health policies. This study aimed to estimate a measure of comparative vaccine effectiveness between Omicron (BA.1) and Delta (B.1.617.2 and sub-lineages) VOC according to vaccination exposure (primary or booster). Methods: We developed a case–case study using data on RT-PCR SARS-CoV2-positive cases notified in Portugal during Weeks 49–51, 2021. To obtain measure of comparative vaccine effectiveness, we compared the odds of vaccination in Omicron cases versus Delta using logistic regression adjusted for age group, sex, region, week of diagnosis, and laboratory of origin. Results: Higher odds of vaccination were observed in cases infected by Omicron VOC compared with Delta VOC cases for both complete primary vaccination (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.8 to 2.4) and booster dose (OR = 5.2; 95% CI: 3.1 to 8.8), equivalent to reduction of vaccine effectiveness from 44.7% and 92.8%, observed against infection with Delta, to 6.0% (95% CI: 29.2% to 12.7%) and 62.7% (95% CI: 35.7% to 77.9%), observed against infection with Omicron, for complete primary vaccination and booster dose, respectively. Conclusion: Consistent reduction in vaccine-induced protection against infection with Omicron was observed. Complete primary vaccination may not be protective against SARS-CoV-2 infection in regions where Omicron variant is dominant.Grant no. 2021/PHF/23776; POCI-01-0145-FEDER-022184; Project ALG-D2-2021-06info:eu-repo/semantics/publishedVersio

Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus

Erratum: Nat Med. 2022 Oct;28(10):2220-2221. doi: 10.1038/s41591-022-02036-2. https://www.nature.com/articles/s41591-022-02036-2The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.info:eu-repo/semantics/publishedVersio