Accelerating SARS-CoV-2 genomic surveillance in a routine clinical setting with nanopore sequencing.

BACKGROUND: SARS-CoV-2 genomic analysis has been key to the provision of valuable data to meet both epidemiological and clinical demands. High-throughput sequencing, generally Illumina-based, has been necessary to ensure the widest coverage in global variant tracking. However, a speedier response is needed for nosocomial outbreak analyses and rapid identification of patients infected by emerging VOCs. An alternative based on nanopore sequencing may be better suited to delivering a faster response when required; however, although there are several studies offering side-by-side comparisons of Illumina and nanopore sequencing, evaluations of the usefulness in the hospital routine of the faster availability of data provided by nanopore are still lacking. RESULTS: We performed a prospective 10-week nanopore-based sequencing in MinION in a routine laboratory setting, including 83 specimens where a faster response time was necessary. The specimens analyzed corresponded to i) international travellers in which lineages were assigned to determine the proper management/special isolation of the patients; ii) nosocomial infections and health-care-worker infections, where SNP-based comparisons were required to rule in/out epidemiological relationships and tailor specific interventions iii) sentinel cases and breakthrough infections to timely report to the Public Health authorities. MinION-based sequencing was compared with the standard procedures, supported on Illumina sequencing; MinION accelerated the delivery of results (anticipating results 1-12 days) and reduced costs per sample by 28€ compared to Illumina, without reducing accuracy in SNP calling. CONCLUSIONS: Parallel integration of Illumina and nanopore sequencing strategies is a suitable solution to ensure both high-throughput and rapid response to cope with accelerating the surveillance demands of SARS-CoV-2 while also maintaining accuracy.

A solution to achieve sequencing from SARS-CoV-2 specimens with low viral loads: concatenation of reads from independent reactions.

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 specimens that involves sequencing in triplicate and concatenation of the reads obtained using bioinformatics. This proposal is based on the hypothesis that the uncovered regions in each replicate differ and that concatenation would compensate for these gaps and recover a larger percentage of the sequenced genome. RESULTS: Whole genome sequencing was performed in triplicate on 30 samples with Ct > 32 and the benefit of replicate read concatenation was assessed. After concatenation: i) 28% of samples reached the standard quality coverage threshold (> 90% genome covered > 30x); ii) 39% of samples did not reach the coverage quality thresholds but coverage improved by more than 40%; and iii) SARS-CoV-2 lineage assignment was possible in 68.7% of samples where it had been impaired. CONCLUSIONS: Concatenation of reads from replicate sequencing reactions provides a simple way to access hidden information in the large proportion of SARS-CoV-2-positive specimens eliminated from analysis in standard sequencing schemes. This approach will enhance our potential to rule out involvement in outbreaks, to characterize reinfections and to identify lineages of concern for surveillance or therapeutical purposes.

Early SARS-CoV-2 Reinfections Involving the Same or Different Genomic Lineages, Spain.

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 a sample of 26 patients with 2 COVID-19 episodes separated by 20-45 days. Among sampled patients, 11 (42%) had reinfections involving different SARS-CoV-2 variants or subvariants. Another 4 cases were probable reinfections; 3 involved different strains from the same lineage or sublineage. Host genomic analysis confirmed the 2 sequential specimens belonged to the same patient. Among all reinfections, 36.4% involved non-Omicron, then Omicron lineages. Early reinfections showed no specific clinical patterns; 45% were among unvaccinated or incompletely vaccinated persons, 27% were among persons <18 years of age, and 64% of patients had no risk factors. Time between sequential positive SARS-CoV-2 PCRs to consider reinfection should be re-evaluated.

A One Health approach revealed the long-term role of Mycobacterium caprae as the hidden cause of human tuberculosis in a region of Spain, 2003 to 2022.

IntroductionMycobacterium caprae is a member of the Mycobacterium tuberculosis complex (MTBC) not routinely identified to species level. It lacks specific clinical features of presentation and may therefore not be identified as the causative agent of tuberculosis. Use of whole genome sequencing (WGS) in the investigation of a family microepidemic of tuberculosis in Almería, Spain, unexpectedly identified the involvement of M. caprae.AimWe aimed to evaluate the presence of additional unidentified M. caprae cases and to determine the magnitude of this occurrence.MethodsFirst-line characterisation of the MTBC isolates was done by MIRU-VNTR, followed by WGS. Human and animal M. caprae isolates were integrated in the analysis.ResultsA comprehensive One Health strategy allowed us to (i) detect other 11 M. caprae infections in humans in a period of 18 years, (ii) systematically analyse M. caprae infections on an epidemiologically related goat farm and (iii) geographically expand the study by including 16 M. caprae isolates from other provinces. Integrative genomic analysis of 41 human and animal M. caprae isolates showed a high diversity of strains. The animal isolates' diversity was compatible with long-term infection, and close genomic relationships existed between isolates from goats on the farm and recent cases of M. caprae infection in humans.DiscussionZoonotic circulation of M. caprae strains had gone unnoticed for 18 years. Systematic characterisation of MTBC at species level and/or extended investigation of the possible sources of exposure in all tuberculosis cases would minimise the risk of overlooking similar zoonotic events.

Association of blood-based biomarkers with radiologic markers and cognitive decline in atrial fibrillation patients.

BACKGROUND: Atrial fibrillation (AF) has been associated with an increased risk of silent brain infarcts (SBI) and cognitive impairment, even in patients with low embolic risk. We aimed to test the association between 11 blood-biomarkers representing different AF-related pathways, and SBI, white matter hyperintensities (WMH), and cognitive decline in patients with AF and low embolic risk. METHODS: The present study followed a cross-sectional design. 70 patients with a history of AF and CHADS2 score ≤1, and 10 controls with neither AF nor SBI were included. All patients underwent a 3T brain MRI. Cortical and large subcortical ischemic lesions were considered presumed embolic origin lesions. White matter hyperintensities (WMH) were measured according to the Fazekas scale. A subset of patients underwent cognitive evaluation with the MoCA test. Circulating proteins were measured under blind conditions in a laboratory at Roche Diagnostics, Germany. RESULTS: 45 patients presented SBI in the MRI, and 25 did not. Ang-2, FGF-23, and BMP-10 were increased in patients with SBI. Ang-2 was elevated only in patients with embolic infarcts, whereas FGF-23 and BMP-10 tended to be elevated in patients with both types of infarcts. Ang-2 (OR = 1.56 [0.94-2.59], p = 0.087), and BMP-10 (OR = 4.83 [0.99-23.60], p = 0.052) were the biomarkers that showed the highest association with SBI when entered in a multivariable logistic regression model corrected by age. No biomarker was found associated with WMH or mild cognitive impairment. CONCLUSIONS: BMP-10, and Ang-2 were increased in patients with SBI. Its usefulness to detect SBI in AF patients should be further explored.

Angiosarcoma of the aorta diagnosed via endoscopic ultrasound.

A 54-year-old man consulted for low back pain of 5 weeks of evolution, refractory to regular analgesics, and significant weight loss. The PET-CT revealed a retroperitoneal mass in contact with the anterior wall of the abdominal aorta. After consulting with the Endoscopy Unit, an endoscopic ultrasound-guided FNAP was performed due to the accessibility of the lesion and the less invasive nature of these procedures. The anatomopathological result was angiosarcoma of the aorta.

Proper Assignation of Reactivation in a COVID-19 Recurrence Initially Interpreted as a Reinfection.

A 77-year-old man (case R) with previous diagnosis of a mild COVID-19 episode was hospitalized 35 days later. On day 23 postadmission, he developed a second COVID-19 episode, now severe, and finally died. Initially, case R's COVID-19 recurrence was interpreted as a reinfection due to the exposure to a SARS-CoV-2 RT-PCR-positive roommate. However, whole-genome sequencing indicated that case R's recurrence corresponded to a reactivation of the strain involved in his first episode. Case R's reactivation had major consequences, leading to a more severe episode, and causing subsequent transmission to another 2 hospitalized patients, 1 of them with fatal outcome.

Sex-related disparities in the incidence and outcomes of hemorrhagic stroke among type 2 diabetes patients: a propensity score matching analysis using the Spanish National Hospital Discharge Database for the period 2016-18.

BACKGROUND: To analyze incidence, use of therapeutic procedures, use of oral anticoagulants (OACs) and antiplatelet agents prior to hospitalization, and in-hospital outcomes among patients who were hospitalized with hemorrhagic stroke (HS) according to the presence of type 2 diabetes mellitus (T2DM) in Spain (2016-2018) and to assess the role of sex differences among those with T2DM. METHODS: Using the Spanish National Hospital Discharge Database we estimated the incidence of HS hospitalizations in men and women aged ≥ 35 years with and without T2DM. Propensity score matching (PSM) was used to compare population subgroups according to sex and the presence of T2DM. RESULTS: HS was coded in 31,425 men and 24,975 women, of whom 11,915 (21.12%) had T2DM. The adjusted incidence of HS was significantly higher in patients with T2DM (both sexes) than in non-T2DM individuals (IRR 1.15; 95% CI 1.12-1.17). The incidence of HS was higher in men with T2DM than in T2DM women (adjusted IRR 1.60; 95% CI 1.57-1.63). After PSM, men and women with T2DM have significantly less frequently received decompressive craniectomy than those without T2DM. In-hospital mortality (IHM) was higher among T2DM women than matched non-T2DM women (32.89% vs 30.83%; p = 0.037), with no differences among men. Decompressive craniectomy was significantly more common in men than in matched women with T2DM (5.81% vs. 3.33%; p < 0.001). IHM was higher among T2DM women than T2DM men (32.89% vs. 28.28%; p < 0.001). After adjusting for confounders with multivariable logistic regression, women with T2DM had a 18% higher mortality risk than T2DM men (OR 1.18; 95% CI 1.07-1.29). Use of OACs and antiplatelet agents prior to hospitalization were associated to higher IHM in men and women with and without T2DM. CONCLUSIONS: T2DM is associated with a higher incidence of HS and with less frequent use of decompressive craniectomy in both sexes, but with higher IHM only among women. Sex differences were detected in T2DM patients who had experienced HS, with higher incidence rates, more frequent decompressive craniectomy, and lower IHM in men than in women.

Are there sex differences in the effect of type 2 diabetes in the incidence and outcomes of myocardial infarction? A matched-pair analysis using hospital discharge data.

BACKGROUND: To analyze incidence, use of therapeutic procedures, and in-hospital outcomes in patients with ST elevation myocardial infarction (STEMI) and non-ST elevation myocardial infarction (NSTEMI) according to the presence of type 2 diabetes (T2DM) in Spain (2016-2018) and to investigate sex differences. METHODS: Using the Spanish National Hospital Discharge Database, we estimated the incidence of myocardial infarctions (MI) in men and women with and without T2DM aged ≥ 40 years. We analyzed comorbidity, procedures, and outcomes. We matched each man and woman with T2DM with a non-T2DM man and woman of identical age, MI code, and year of hospitalization. Propensity score matching was used to compare men and women with T2DM. RESULTS: MI was coded in 109,759 men and 44,589 women (30.47% with T2DM). The adjusted incidence of STEMI (IRR 2.32; 95% CI 2.28-2.36) and NSTEMI (IRR 2.91; 95% CI 2.88-2.94) was higher in T2DM than non-T2DM patients, with higher IRRs for NSTEMI in both sexes. The incidence of STEMI and NSTEMI was higher in men with T2DM than in women with T2DM. After matching, percutaneous coronary intervention (PCI) was less frequent among T2DM men than non-T2DM men who had STEMI and NSTEMI. Women with T2DM and STEMI less frequently had a code for PCI that matched that of non-T2DM women. In-hospital mortality (IHM) was higher among T2DM women with STEMI and NSTEMI than in matched non-T2DM women. In men, IHM was higher only for NSTEMI. Propensity score matching showed higher use of PCI and coronary artery bypass graft and lower IHM among men with T2DM than women with T2DM for both STEMI and NSTEMI. CONCLUSIONS: T2DM is associated with a higher incidence of STEMI and NSTEMI in both sexes. Men with T2DM had higher incidence rates of STEMI and NSTEMI than women with T2DM. Having T2DM increased the risk of IHM after STEMI and NSTEMI among women and among men only for NSTEMI. PCI appears to be less frequently used in T2DM patients After STEMI and NSTEMI, women with T2DM less frequently undergo revascularization procedures and have a higher mortality risk than T2DM men.

The influence of sex on ischemic stroke incidence, therapeutic procedures and in-hospital mortality: Results of the Spanish National Hospital Discharge.

BACKGROUND: Previous research has revealed sex-related differences in outcomes for people admitted to hospitals for ischemic stroke. We aimed to analyse the incidence, use of invasive procedures and in-hospital outcomes of ischemic stroke in Spain (2016-2018) using the Spanish National Hospital Discharge Database. We sought sex-related differences in incidence and in-hospital outcomes over time. METHODS: We estimated the incidence of ischemic stroke in men and women. We analysed comorbidities (Charlson's comorbidity index, cardiovascular risk factors, alcohol abuse and atrial fibrillation), procedures (mechanical ventilation, endovascular thrombectomy and thrombolytic therapy) and outcomes. We matched each woman with a man with identical age, type of ischemic stroke and year of hospitalisation. We built Poisson regression models to obtain adjusted incidence rate ratios (IRRs). We tested in-hospital mortality (IHM) with logistic regression analyses. RESULTS: Ischemic stroke was coded in 172 255 patients aged ≥35 years (92 524 men 53.7%). Men showed higher incidence rates (216.9 vs. 172.3/105 ; P < .001; IRR = 1.57 (95% CI:1.55-1.59) than women. After matching, the use of endovascular thrombectomy (5.1% vs. 4.0%; P < .001) and thrombolytic therapy (7.6% vs. 6.8%; P < .001) was higher among women. IHM was significantly higher in women than in matched men (11.2% vs. 10.4%; P < .001). Women had a lower IHM than matched men when endovascular thrombectomy (9.4% vs. 12.1%; P = .001) or thrombolytic therapy (6.7% vs. 8.3%; P = .003) was coded. Patients of both sexes admitted for ischemic stroke who received thrombolytic therapy had lower IHM (OR = 0.76; 95% CI:0.68-0.85 among men; and OR = 0.58; 95% CI:0.52-0.64 among women), but endovascular thrombectomy was associated with a lower IHM only among women (OR = 0.58; 95% CI:0.51-0.66). After multivariable adjusting, women admitted to the hospital for ischemic stroke had a significantly higher IHM than men (OR = 1.16; 95% CI:1.12-1.21). CONCLUSION: Men had higher incidence rates of ischemic stroke than women. Women more often underwent thrombolytic therapy and endovascular thrombectomy but had a higher IHM.

The significance of heart failure in hospitalised patients with pulmonary embolism. A gender-specific analysis.

BACKGROUND: Heart failure (HF) is a risk factor for the development of pulmonary embolism (PE). Few studies have examined sex differences in risk of PE among HF patients. AIMS: (a) To examine the incidence, characteristics and in-hospital outcomes among patients hospitalised with PE according to HF status; (b) to compare the in-hospital mortality (IHM) after PE between HF and non-HF patients and (c) to identify variables associated with IHM. All analyses were stratified by sex. METHODS: We included all adult patients hospitalised for PE from 1 January 2016 to 31 December 2018. Data were collected from the Spanish National Hospital Discharge Database. Poisson regression models were constructed to quantify the difference in the incidences between HF and non-HF populations. Propensity score matching (PSM) was used to obtain comparable subgroups by sex and HF status. RESULTS: We identified 46,835 PE hospitalisations, 11.4% with HF. Adjusted incidence of PE was higher in HF patients than in those without HF (Incidence Rate Ratio 1.11; 95% CI 1.08-1.13). Crude and PSM adjusted IHM were significantly higher in men and women hospitalised with PE suffering HF than in men and women without HF (P < .001). Women with HF who suffered a PE had lower IHM than men with this condition (P < .001) after adjusting. CONCLUSIONS: Adjusted incidence of PE was higher in HF patients than in those without HF. After PSM suffering, HF was associated to higher IHM in men and women. Women with PE and HF had lower IHM than men with these conditions.

Gender differences in incidence and in-hospital outcomes of community-acquired, ventilator-associated and nonventilator hospital-acquired pneumonia in Spain.

AIMS: We aim to compare the incidence and in-hospital outcomes of community-acquired pneumonia (CAP), ventilator-associated pneumonia (VAP) and nonventilator hospital-acquired pneumonia (NV-HAP) according to gender. METHODS: This was a retrospective observational epidemiological study using the Spanish National Hospital Discharge Database for the years 2016 and 2017. RESULTS: Of 277 785 hospital admissions, CAP was identified in 257 455 (41.04% females), VAP was identified in 3261 (30.42% females) and NV-HAP was identified in 17 069 (36.58% females). The incidence of all types of pneumonia was higher amongst males (CAP: incidence rate ratio [IRR] 1.05, 95% CI 1.03-1.06; VAP: IRR 1.36, 95% CI 1.26-1.46; and NV-HAP: IRR 1.16, 95% CI 1.14-1.18). The crude in-hospital mortality (IHM) rate for CAP was 11.44% in females and 11.80% in males (P = .005); for VAP IHM, the rate was approximately 35% in patients of both genders and for NV-HAP IHM, the rate was 23.97% for females and 26.40% for males (P < .001). After multivariable adjustment, in patients of both genders, older age and comorbidities were factors associated with IHM in the three types of pneumonia analysed. Female gender was a risk factor for IHM after VAP (OR 1.24; 95% CI 1.06-1.44), and no gender differences were found for CAP or NV-HAP. CONCLUSIONS: Our findings show a difference between females and males, with females presenting a lower incidence of all types of pneumonia. However, female gender was a risk factor for IHM after VAP.

Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru.

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-resistant tuberculosis globally.

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

BackgroundThe 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 surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

The use of serum calprotectin as a biomarker for inflammatory activity in inflammatory bowel disease.

INTRODUCTION: simple, reliable and non-invasive biomarkers are needed to enable the early detection of inflammatory activity for the correct management of inflammatory bowel disease (IBD). One of these biomarkers may be serum calprotectin (SC). MATERIAL AND METHODS: a prospective study was performed of patients with IBD due to undergo a colonoscopy as part of the common clinical practice. The study parameters included SC, fecal calprotectin (FC) and conventional blood test parameters. Clinical indices (Harvey and Walmsley) and relevant endoscopic scores were completed for each scenario (Simple Endoscopic Score Crohn Disease [SES-CD] and Mayo). RESULTS: fifty-three patients were included in the study, 51% (27 patients) with ulcerative colitis (UC) and 49% (26 patients) with Crohn's disease (CD). The CS values in UC were significantly higher with an endoscopic Mayo score 2/3 (median score 10.39 mg/ml [IQR: 7.4-12.2]) compared to those with a Mayo score of 0/1 (median 4.07 mg/ml [IQR: 2.9-7.2]) (p = 0.01). The area under the ROC curve (AUCROC) was 0.85 and the sensitivity and specificity were 83.3% and 81.25%, respectively, for a SC cut-off point of 4.4 mg/dl. Furthermore, a higher AUCROC was obtained in comparison with other serological markers for activity (C-reactive protein [CRP], erythrocyte sedimentation rate [ESR], hemoglobin [Hb] and platelets). There were no statistically significant differences in the comparison between SC and endoscopic findings in CD (SES CD > 3: 20.1 [IQR: 16.8-23.4] vs SESC ≤ 3:6.25 [IQR: 5.4-7.1]) (p = 0.8). CONCLUSIONS: SC is a good indirect marker of inflammatory activity and there was a correlation with endoscopic findings in UC. However, there were no statistically significant differences in the case of CD.

Clonal Complexity in Mycobacterium tuberculosis Can Hamper Diagnostic Procedures.

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 when the susceptibility patterns of the strains differ. Here, we show how mixed infections can also prove challenging for other diagnostic procedures, even outside settings where mixed infections are traditionally expected. We show how an unnoticed mixed infection in an HIV-positive patient diagnosed in Madrid, Spain, with differences in the representativeness of the coinfecting strains in different sputum samples, markedly complicated the resolution of a laboratory cross-contamination false positivity alert.

Reliability of blood test results in samples obtained using a 2-mL discard volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient.

BACKGROUND: Intensive care patients require a high frequency of blood testing, which results in a significant amount of blood loss. When blood is obtained from a central venous catheter (CVC), a large volume is usually discarded to obtain an unaltered sample for testing. AIM: To determine the reliability of complete blood test results in blood samples obtained from the proximal lumen of a triple-lumen CVC using a 2-mL discard volume DESIGN: Observational study with the prospective collection of data METHODS: The subjects enrolled were all patients with a subclavian triple-lumen CVC, older than 17 years and consecutively admitted to intensive care over a 2-year period. In each of the 54 participants, one blood sample was drawn from the proximal lumen of the catheter, discarding 1·61 mL of blood plus 0·39 mL of catheter deadspace (2 mL) and without interrupting infusion in the middle and distal lumens. A second sample was then obtained by direct venous puncture. The reliability of blood test results was determined by comparing sets of variables recorded for the two sampling methods through intraclass correlation coefficients in the Bland-Altman method. RESULTS: Inter-method reliability for the variables examined was excellent, >0·75; range (0·868-0·998). Mean differences between the two sample types for the variables most often determined in critically ill patients were leukocytes: 0·200 × 103 /µL, 95% confidence interval (CI) (0·025 to 0·375); erythrocytes: 0·045 × 106 /uL, 95% CI (-0·003 to 0·094); sodium: 0·074 mEq/L, 95% CI (-0·369 to 0·517); potassium: -0·002mEq/L, 95% CI (-0·065 to 0·061) and glucose: 2·426 mg/dL, 95% CI (0·498-4·354). CONCLUSIONS: The sampling method proposed minimizes blood loss while offering reliable blood test results. RELEVANCE TO CLINICAL PRACTICE: The main benefit of the method proposed is reduced blood loss, improving the care of a critically ill patient.

Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population.

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 method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission-despite opportunities-and challenged some of the advantages associated with Beijing strains.

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis.

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

Subtle genotypic changes can be observed soon after diagnosis in Mycobacterium tuberculosis infection.

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 (61-85days). Most harbored SNPs and for some of them a potential functional role was suggested. Microevolution in tuberculosis seems to occur sooner and more often than expected and could affect tracking of transmission.