Laura Perez-Lago

Hospital General Universitario Gregorio Marañón | HGGM · Department of Clinical Microbiology

Background During the pandemic, whole genome sequencing was critical to characterize SARS-CoV-2 for surveillance, clinical and therapeutical purposes. However, low viral loads in specimens often led to suboptimal sequencing, making lineage assignment and phylogenetic analysis difficult. We propose an alternative approach to sequencing these specime...

A 77-year-old male (Case R) who had had a previous diagnosis of mild COVID-19 episode, with fever without developing pneumonia or other complications, was hospitalized 57 days later, due to an acute cholangitis. He had a prolonged hospital stay with severe biliary and infectious complications. On Day 23 post-admission, the patient developed a secon...

During the COVID-19 pandemic, several SARS-CoV-2 variants of concern (VOCs) of particular relevance emerged. Early detection of VOCs entering a country is essential to control spread. The alert triggered by the first suspected case of the Omicron variant in Spain in a traveler arriving from South Africa in November 2021 provided a unique opportunit...

Background: SARS-CoV-2 recombinants involving the divergent Delta and Omicron lineages have been described, and one of them, "Kraken" (XBB.1.5), has recently been a matter of concern. Recombination requires the coexistence of two SARS-CoV-2 strains in the same individual. Only a limited number of studies have focused on the identification of co-in...

Background: COVID-19 diagnosis lies on the detection of SARS-CoV-2 on nasopharyngeal specimens by RT-PCR. The Xpert-Xpress SARS-CoV-2 assay provides results in less than one hour from specimen reception, which makes it suitable for clinical/epidemiological circumstances that require faster responses. The analysis of a COVID-19 outbreak suspected i...

The emergence of the Omicron variant of SARS-CoV-2 represented a challenge to the treatment of COVID-19 using monoclonal antibodies. Only Sotrovimab maintained partial activity, allowing it to be used in high-risk patients infected with the Omicron variant. However, reports of resistance mutations to Sotrovimab demand efforts to better understand t...

Centers for Disease Control and Prevention guidelines consider SARS-CoV-2 reinfection when sequential COVID-19 episodes occur >90 days apart. However, genomic diversity acquired over recent COVID-19 waves could mean previous infection provides insufficient cross-protection. We used genomic analysis to assess the percentage of early reinfections in...

Outbreak strains of Mycobacterium tuberculosis are promising candidates as targets in the search for intrinsic determinants of transmissibility, as they are responsible for many cases with sustained transmission; however, the use of low-resolution typing methods and restricted geographical investigations represent flaws in assessing the success of...

Despite the proven value of applying genomic data for epidemiological purposes, commonly used high-throughput sequencing formats are not adapted to the response times required to intervene and finally control outbreaks. In this study, we propose a fast alternative to whole-genome sequencing (WGS) to track relevant microbiological strains: nanopore...

Introduction A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and methods We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caus...

A monkeypox virus (MPXV) outbreak has been ongoing worldwide since May 2022. The role of specimens other than skin lesions for MPXV diagnosis is unknown. We evaluated 140 different clinical specimens by real-time PCR. The highest positivity rates (97%) were from skin lesions of any part of the body, followed by plasma, pharyngeal and anal swabs. Te...

Introduction SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. Methods Whole genome sequencing was performed prospective...

Genotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spa...

COVID-19 vaccination has proven to be effective at preventing symptomatic disease but there are scarce data to fully understand whether vaccinated individuals can still behave as SARS-CoV-2 transmission vectors. Based on viral genome sequencing and detailed epidemiological interviews, we report a nosocomial transmission event involving two vaccinat...

We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most app...

Estimates of the burden of severe acute respiratory syndrome coronavirus 2 reinfections are limited by the scarcity of population-level studies incorporating genomic support. We conducted a systematic study of reinfections in Madrid, Spain, supported by genomic viral analysis and host genetic analysis, to cleanse laboratory errors and to discrimina...

The SARS-CoV-2 variant of concern (VOC) Delta (B.617.2 lineage) displaced the predominant VOC Alpha (B.1.1.7 lineage) in the United Kingdom. In Madrid, recent start of the decline of predominant VOC Alpha suggested an equivalent phenomenon. However, 11 different variants, none overrepresented in frequency, occupied progressively over a period of 7...

196 travellers with negative-COVID-19-tests prior to departure tested positive on arrival at Madrid (April/June-2021), from a total of 45.211 travellers tested (0.43%). Viral loads (Ct 20.3), were higher compared to the general population (Ct 27.09). Our data reveal weaknesses in pre-departure testing and alert about high-viral-load-SARS-CoV-2 carr...

Reverse transcription-PCRs (RT-PCRs) targeting SARS-CoV-2 variant of concern (VOC) mutations have been developed to simplify their tracking. We evaluated an assay targeting E484K/N501Y to identify B.1.351/P1. Whole-genome sequencing (WGS) confirmed only 72 (59.02%) of 122 consecutive RT-PCR P.1/B.1.351 candidates. Prescreening RT-PCRs must target a...

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identifi...

We report a COVID‐19 case with unprecedented viral complexity. In the first severe episode, two different SARS‐CoV‐2 strains (superinfection) were identified within a week. Three months after discharge, patient was readmitted and was infected in a nosocomial outbreak with a different strain, suffering a second milder COVID‐19 episode. This article...

SARS-CoV-2 nosocomial outbreaks in the first COVID-19 wave were likely associated with a shortage of personal protective equipment and scarce indications on control measures. Having covered these limitations, updates on current SARS-CoV-2 nosocomial outbreaks are required. We carried out an in-depth analysis of a 27-day nosocomial outbreak in a gas...

Following its emergence in late 2019, the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1,2 has been tracked via phylogenetic analysis of viral genome sequences in unprecedented detail3–5. While the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced. However, wit...

A successful Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variant, B.1.1.7, has recently been reported in the UK, causing global alarm. Most likely, the new variant emerged in a persistently infected patient, justifying a special focus on these cases. Our aim in this study was to explore certain clinical profiles involving severe im...

We report a COVID-19 case with unprecedented viral complexity. In the first severe episode, two different SARS-CoV-2 strains (superinfection) were identified within a week. Three months after discharge, patient was readmitted and was infected in a nosocomial outbreak with a different strain, suffering a second milder COVID-19 episode.

Introduction: SARS-CoV-2 variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. Methods: Whole genome sequencing was performed prospecti...

Objectives To characterize the clonal complexity in Mycobacterium tuberculosis (MTB) infections considering factors that help maximize the detection of coexisting strains/variants. Methods Genotypic analysis by Mycobacterial Interspersed Repetitive-Unit - Variable-Number Tandem-Repeats (MIRU-VNTR) was performed directly on 70 biopsy specimens from...

Background: Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy’s decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed...

We present clinical, genomic and epidemiological data on the first 106 cases with the SARS-CoV-2 B.1.1.7 variant in Madrid. Even from the start, the increase of this variant was due to transmission events within the community, some causing extensive clusters, rather than further imports. Most cases developed non-severe disease.

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypas...

SARS-CoV-2 nosocomial outbreaks in the first COVID-19 wave were likely associated to a shortage of personal protective equipment and scare indications on control measures. Having covered these limitations, updates on current SARS-CoV-2 nosocomial outbreaks are required. We carried out an in-depth analysis of a 27-day nosocomial outbreak in a gastro...

Introduction: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and Methods: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, cau...

SARS‐CoV2‐RT‐PCR cycle‐threshold values from 18,803 cases (2 March‐4 October) in Madrid define three stages: i) initial ten weeks with sustained reduction of viral load (Ct: 23.4‐32.3), ii) stability with low‐viral loads (Ct:31.9‐35.5) in the next nine weeks, and iii) sudden increase with progressive higher viral loads until reaching stability at h...

Objectives: The purpose of this study was to detect COVID-19 cases with persistent positive RT-PCR results for SARS-CoV-2, for which viable virus can be inferred, due to the presence of subgenomic (SG) viral RNA, which is expressed only in replicating viruses. Methods: RNA remnants, purified from diagnostic nasopharyngeal specimens, were used as te...

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analyzed 90 sequential MDR-Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR-TB that recurred in 2018 (1-10 years, 2-10 isolates per patient). Three long-term predominant strains were responsible for 63% of all MDR-TB recurrenc...

The COVID-19 pandemic has shaken the world since the beginning of 2020. Spain is among the European countries with the highest incidence of the disease during the first pandemic wave. We established a multidisciplinar consortium to monitor and study the evolution of the epidemic, with the aim of contributing to decision making and stopping rapid sp...

Beijing genotype Mycobacterium tuberculosis strains associate with increased virulence, resistance and/or higher transmission rates. This study describes a specific Beijing strain predominantly identified in the Panamanian province of Colon with one of the highest incidences of tuberculosis in the country. Retrospective mycobacterial interspersed r...

An outbreak involving 14 patients infected by an extensively drug resistance Pseudomonas aeruginosa strain was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy to investigate in-depth the outbreak by means of molecular and genomic approaches. Pulsed-Field-Gel-Electrophoresis was applied as a first-line...

Two Mycobacterium tuberculosis strains-M (sublineage 4.1) and Ra (sublineage 4.3)-have long prevailed in Argentina among patients with multidrug-resistant tuberculosis (MDR-TB). Recently, budget constraints have hampered the surveillance of MDR-TB transmission. Based on whole-genome sequence analysis, we used M- and Ra-specific single nucleotide po...

Transmission of Beijing Mycobacterium tuberculosis can be investigated based on genotypic analysis of clinical isolates. A Beijing strain began to spread on Gran Canaria Island, Spain, at the end of the last century. In 1996, only 3 years after its importation to the island, its frequency had increased to 27.1% of all the isolates. The strain was t...

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates incl...

We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-re...

Background We present our experience in patients with hematologic malignancy and Pseudomonas aeruginosa infection treated with ceftolozane-tazobactam. Materials/methods We performed a single-center case-control study including all patients with hematologic malignancy and P. aeruginosa infection treated with ceftolozane-tazobactam (study group) and...

Background The analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillan...

The Evolution of Genotyping Strategies To Detect, Analyze, and Control Transmission of Tuberculosis, Page 1 of 2 Abstract The introduction of genotypic tools to analyze Mycobacterium tuberculosis isolates has transformed our knowledge of the transmission dynamics of this pathogen. We discuss the development of the laboratory methods that have bee...

Mycobacterium chimaera is involved in a worldwide-alert owing to contaminated heater-cooler-units. We implemented an RT-PCR-based procedure to survey undetected cases of M. chimaera infection. The PCR was negative in the 59 prosthetic heart-valves from patients with PCR-16SrRNA-negative infective endocarditis. PCR identified M. chimaera in one of 1...

Background:Mycobacterium tuberculosis (MTB) has limited ability to acquire variability. Analysis of its microevolution might help us to evaluate the pathways followed to acquire greater infective success. Whole-genome sequencing (WGS) in the analysis of the transmission of MTB has elucidated the magnitude of variability in MTB. Analysis of transmis...

SNPs and features for the SNPs found for the intrapatient analysis.

SNPs and features for the SNPs found for the Cluster 6.

SNPs and features for the SNPs found for the Cluster B.

SNPs and features for the SNPs found for the Cluster F.

SNPs and features for the SNPs found for the Clusters 1, 2 and 3.

The assignation of lineages in Mycobacterium tuberculosis (MTB) provides valuable information for evolutionary and phylogeographic studies and makes for more accurate knowledge of the distribution of this pathogen worldwide. Differences in virulence have also been found for certain lineages. MTB isolates were initially assigned to lineages based on...

Purpose: Genotypic methods have considerably improved the diagnosis of multidrug-resistant (MDR) tuberculosis. One of these tests is Anyplex II MTB/MDR/XDR (Anyplex). Our aim was to evaluate the diagnostic performance of this multiplex PCR. Methods: We conducted our study on 47 MDR tuberculosis and 14 pan-susceptible strains. We evaluated the ab...

Clonal complexity is increasingly accepted in Mycobacterium tuberculosis infection, including mixed infections by ≥2 strains, which usually occur in settings with a high burden of tuberculosis and/or a high risk of overexposure to infected patients. Mixed infections can hamper diagnostic procedures: obtaining an accurate antibiogram is difficult wh...

The role of clonal complexity has gradually been accepted in infection by Mycobacterium tuberculosis (MTB), although analyses of this issue are limited. We performed an in-depth study of a case of recurrent MTB infection by integrating genotyping, whole genome sequencing, analysis of gene expression and infectivity in in vitro and in vivo models. F...

Molecular epidemiology analysis of tuberculosis transmission is based mostly on the application of MIRU-VNTR. In certain isolates a complete 24-loci genotype is not obtained and these incompletely genotyped isolates can not be used in the definition of clusters. In a population-based molecular epidemiology study performed in Almería, Southeast Spai...

The national tuberculosis programme (NTP) of Cameroon has recently reported that an alarming number of tuberculosis (TB) patients are border-crossing from neighboring Equatorial Guinea to seek multi-drug-resistant tuberculosis (MDR-TB) treatment [1, 2]. The World Health Organization (WHO) African Region is reported to bear a low MDR-TB proportion o...

Current migratory movements require new strategies for rapid tracking of the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design polymerase-chain-reaction assays (PCRs) to track specific relevant strains. However, fast...

Objective: Molecular epidemiology techniques in tuberculosis (TB) can identify high-risk strains that are actively transmitted. We aimed to implement a novel strategy to optimize the identification and control of MDR-TB in a specific population. Methods: We developed a strain-specific-PCR tailored from whole-genome-sequencing (WGS) data to track...

Clonal variants of Mycobacterium tuberculosis (MTB) coexist in specific patients, although the dynamics of their emergence is unknown. We used MIRU-VNTR to detect microevolution leading to variants of MTB in 3 out of 19 patients (15%) soon after diagnosis (61-85 days). Most harbored SNPs and for some of them a potential functional role was suggeste...

A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR based on whole-genome sequencing to target strain-specific SNPs. In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immig...

In vitro fitness of the multidrug-resistant and susceptible Mycobacterium tuberculosis strains involved in the mixed infection.

The strains involved in tuberculosis outbreaks are considered to be highly virulent and transmissible. We analyzed the case of a patient in Madrid, Spain who was persistently infected over an 8-year period by the same Beijing Mycobacterium tuberculosis (MTB) strain. The strain was responsible for a severe outbreak on Gran Canaria Island. The case p...

Molecular epidemiology has transformed our knowledge of how tuberculosis (TB) is transmitted. Whole genome sequencing (WGS) has reached unprecedented levels of accuracy. However, it has increased technical requirements and costs, and analysis of data delays results. Our objective was to find a way to reconcile speed and ease of implementation with...

The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the c...

Mycobacterium bovis is both the causative agent of bovine tuberculosis (TB) and a zoonotic pathogen. In humans, considerably fewer cases of TB are caused by M. bovis than M. tuberculosis; nevertheless, diagnostic limitations mean that currently available data on prevalence grossly underestimate the true dimension of the problem. The routes of trans...

Clonal variants of Mycobacterium tuberculosis can emerge as a result of microevolution in a single host or after sequential infection of different hosts. The significance of subtle genotypic variations is still unknown. In three of the four loci analyzed from clonal variants differing in only one MIRU-VNTR locus, we found that the expression of the...

Clonal variants of Mycobacterium tuberculosis can emerge as a result of microevolution phenomena. The functional significance of these subtle genetic rearrangements is normally disregarded. We show that clonal variants from two patients had different infective behaviours in some in vitro cellular infection models but not in others. Microevolution m...

Background: It has been accepted that the infection by Mycobacterium tuberculosis (M. tuberculosis) can be more heterogeneous than considered. The emergence of clonal variants caused by microevolution events leading to population heterogeneity is a phenomenon largely unexplored. Until now, we could only superficially analyze this phenomenon by sta...

Microevolved Mycobacterium tuberculosis clonal variants from a parental strain can emerge within a single patient infection and during transmission events. Genotypic rearrangements may involve functional changes conferring advantages to favor strain adaptation to the host. In the present study, we analyzed in depth some genotypic characteristics of...

A bacteriophage Ø29 transcriptional regulator, protein p4, interacts with its DNA target by employing two mechanisms: by direct readout of the chemical signatures of only one DNA base and by inducing local modification on the topology of short A tracts (indirect readout). p4 binds as a dimer to targets consisting of imperfect inverted repeats. Here...

We performed an in-depth analysis of a large Mycobacterium tuberculosis cluster involving nine isolates with identical or highly similar RFLP types. The strain involved in this transmission cluster microevolved and accumulated up to five differential IS6110 transposition events, several of them not revealed by standard genotyping approaches. Some o...

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal varian...

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied...

Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions....

Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher prefe...

The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4–DNA complex contains two identical protomers that make similar interactions with the target sequence 5′-AACTTTTT-15 bp-AAAATGTT-3′. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 bindi...

Protein p4 affects the transcriptional switch that divides bacteriophage phi29 infection in early and late phases. The synthesis of DNA replication proteins and p4 takes place in the early phase, while structural, morphogenesis, and lysis proteins are synthesized in the late phase. Transcriptional switch by p4 is achieved by activating the late pro...

Bacteriophage φ29 protein p4 is essential for the regulation of the switch from early to late phage transcription. The protein binds to two regions of the phage genome located between the regulated promoters. Each region contains two inverted repeats separated by 1 bp. We used circular permutation assays to study the topology of the DNA upon bindin...

Transcription regulation relies on the molecular interplay between the RNA polymerase and regulatory factors. Phages of the φ29-like genus encode two regulatory proteins, p4 and p6. In φ29, the switch from early to late transcription is based on the synergistic binding of proteins p4 and p6 to the promoter sequence, resulting in a nucleosome-like s...