Role of the first WHO mutation catalogue in the diagnosis of antibiotic resistance in Mycobacterium tuberculosis in the Valencia Region, Spain: a retrospective genomic analysis.

BACKGROUND: In June, 2021, WHO published the most complete catalogue to date of resistance-conferring mutations in Mycobacterium tuberculosis. Here, we aimed to assess the performance of genome-based antimicrobial resistance prediction using the catalogue and its potential for improving diagnostics in a real low-burden setting. METHODS: In this retrospective population-based genomic study M tuberculosis isolates were collected from 25 clinical laboratories in the low-burden setting of the Valencia Region, Spain. Culture-positive tuberculosis cases reported by regional public health authorities between Jan 1, 2014, and Dec 31, 2016, were included. The drug resistance profiles of these isolates were predicted by the genomic identification, via whole-genome sequencing (WGS), of the high-confidence resistance-causing variants included in the catalogue and compared with the phenotype. We determined the minimum inhibitory concentration (MIC) of the isolates with discordant resistance profiles using the resazurin microtitre assay. FINDINGS: WGS was performed on 785 M tuberculosis complex culture-positive isolates, and the WGS resistance prediction sensitivities were: 85·4% (95% CI 70·8-94·4) for isoniazid, 73·3% (44·9-92·2) for rifampicin, 50·0% (21·1-78·9) for ethambutol, and 57·1% (34·0-78·2) for pyrazinamide; all specificities were more than 99·6%. Sensitivity values were lower than previously reported, but the overall pan-susceptibility accuracy was 96·4%. Genotypic analysis revealed that four phenotypically susceptible isolates carried mutations (rpoB Leu430Pro and rpoB Ile491Phe for rifampicin and fabG1 Leu203Leu for isoniazid) known to give borderline resistance in standard phenotypic tests. Additionally, we identified three putative resistance-associated mutations (inhA Ser94Ala, katG Leu48Pro, and katG Gly273Arg for isoniazid) in samples with substantially higher MICs than those of susceptible isolates. Combining both genomic and phenotypic data, in accordance with the WHO diagnostic guidelines, we could detect two new multidrug-resistant cases. Additionally, we detected 11 (1·6%) of 706 isolates to be monoresistant to fluoroquinolone, which had been previously undetected. INTERPRETATION: We showed that the WHO catalogue enables the detection of resistant cases missed in phenotypic testing in a low-burden region, thus allowing for better patient-tailored treatment. We also identified mutations not included in the catalogue, relevant at the local level. Evidence from this study, together with future updates of the catalogue, will probably lead in the future to the partial replacement of culture testing with WGS-based drug susceptibility testing in our setting. FUNDING: European Research Council and the Spanish Ministerio de Ciencia.

Low-Cost Electronic Nose for the Determination of Urinary Infections.

Currently, urine samples for bacterial or fungal infections require a long diagnostic period (48 h). In the present work, a point-of-care device known as an electronic nose (eNose) has been designed based on the "smell print" of infections, since each one emits various volatile organic compounds (VOC) that can be registered by the electronic systems of the device and recognized in a very short time. Urine samples were analyzed in parallel using urine culture and eNose technology. A total of 203 urine samples were analyzed, of which 106 were infected and 97 were not infected. A principal component analysis (PCA) was performed using these data. The algorithm was initially capable of correctly classifying 49% of the total samples. By using SVM-based models, it is possible to improve the accuracy of the classification up to 74% when randomly using 85% of the data for training and 15% for validation. The model is evaluated as having a correct classification rate of 74%. In conclusion, the diagnostic accuracy of the eNose in urine samples is high, promising and amenable for further improvement, and the eNose has the potential to become a feasible, reproducible, low-cost and high-precision device to be applied in clinical practice for the diagnosis of urinary tract infections.

Population-based sequencing of Mycobacterium tuberculosis reveals how current population dynamics are shaped by past epidemics.

Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application. Here, we generate a population-based dataset from Valencia region and compare it with available datasets from different TB-burden settings to reveal transmission dynamics heterogeneity and its public health implications. We sequenced the whole genome of 785 Mycobacterium tuberculosis strains and linked genomes to patient epidemiological data. We use a pairwise distance clustering approach and phylodynamic methods to characterize transmission events over the last 150 years, in different TB-burden regions. Our results underscore significant differences in transmission between low-burden TB settings, i.e., clustering in Valencia region is higher (47.4%) than in Oxfordshire (27%), and similar to a high-burden area as Malawi (49.8%). By modeling times of the transmission links, we observed that settings with high transmission rate are associated with decades of uninterrupted transmission, irrespective of burden. Together, our results reveal that burden and transmission are not necessarily linked due to the role of past epidemics in the ongoing TB incidence, and highlight the need for in-depth characterization of transmission dynamics and specifically tailored TB control strategies.

Clinical evaluation of a new molecular method for the detection of multidrug-resistant microorganisms.

INTRODUCTION: The main objective of this work is to carry out the clinical validation of the trial with the AMR Direct Flow Chip starting from either nasal swabs, rectal swabs directly or from isolated strains to detect antibiotic resistance genes. METHODS: We developed the preclinical validation of the assay with 104 known bacterial isolates. A total of 210 nasal or rectal swab samples were analyzed. The AMR assay is based on multiplex PCR followed by reverse dot blot hybridization on DNA arrays fully automated by using the HS24 platform. RESULTS: Both the sensitivity and specificity of the preclinical assay were 100%, with the 104 samples correctly identified. In the clinical validation, the sensitivity was 100% and the specificity was between 100% in nasal swabs and 97% in rectal swabs. CONCLUSIONS: The AMR Direct Flow Chip® is a rapid and effective assay for the detection of multidrug-resistant microorganisms (MDR) from nasal and rectal swab samples.

Survival benefit of remdesivir in hospitalized COVID-19 patients with high SARS-CoV-2 viral loads and low-grade systemic inflammation.

OBJECTIVES: To assess the benefits of remdesivir in hospitalized COVID-19 patients receiving combined immunomodulatory therapy (CIT) with dexamethasone and tocilizumab. METHODS: This was a cohort study of microbiologically confirmed COVID-19 hospitalized patients. The primary outcome was all-cause 28 day mortality. Secondary outcomes were need for invasive mechanical ventilation (IMV) and IMV/death. Subgroup analyses according to SARS-CoV-2 cycle threshold (Ct) values and inflammation biomarkers were performed. Multivariable marginal structural Cox proportional hazards regression models were used to analyse the association between remdesivir therapy and the risk of outcomes of interest. RESULTS: Of 1368 hospitalized patients treated with corticosteroids, 1014 (74%) also received tocilizumab, 866 (63%) remdesivir and 767 (56%) tocilizumab + remdesivir. The 28 day mortality was 9% in the overall cohort, with an adjusted HR (aHR) of 0.32 (95% CI = 0.17-0.59) for patients receiving CIT. In the latter group, the 28 day mortality was 6.5%, with an aHR of 1.11 (95% CI = 0.57-2.16) for remdesivir use and there were no differences in secondary outcomes. The risk of primary and secondary outcomes with remdesivir differed by Ct and C-reactive protein (CRP) levels in patients receiving CIT: for 28 day mortality, the aHR was 0.48 (95% CI = 0.21-1.11) for Ct <25, 0.12 (95% CI = 0.02-0.66) for Ct <25 and <5 day symptom duration and 0.13 (95% CI = 0.03-0.50) for CRP <38 mg/L; for IMV and IMV/death, the aHR was 0.32 (95% CI = 0.13-0.77) and 0.33 (95% CI = 0.17-0.63), respectively, in patients with Ct <25. CONCLUSIONS: The benefits of remdesivir administered with dexamethasone and tocilizumab in hospitalized COVID-19 patients differ depending on Ct and CRP. Remdesivir decreases the risk of mortality and need for IMV in patients with high viral loads and low-grade systemic inflammation.

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

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 identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Late reinfection with a different severe acute respiratory syndrome coronavirus-2 clade in a patient with refractory arterial hypertension: a case report.

BACKGROUND: Differentiating between persistent infection with intermittent viral shedding and reinfection with severe acute respiratory syndrome coronavirus 2 remains challenging. Although a small number of cases with genomic evidence of second infection have been reported, limited information exists on frequency and determinants of reinfection, time between infections, and duration of immunity after the primary infection. CASE PRESENTATION: We report a reinfection with severe acute respiratory syndrome coronavirus 2 in a 52-year-old caucasian male whose primary infection was diagnosed in May 2020, during the first wave of the pandemic in Spain, and the second occurred 8 months later, in January 2021. We present a complete dataset including results from real-time polymerase chain reaction, serology, and genome sequencing confirming reinfection with a different clade. Noteworthy was that the patient was immunocompetent but had multiple cardiometabolic comorbidities, including refractory arterial hypertension, that might increase the individual risk in coronavirus disease 2019. CONCLUSIONS: This case of reinfection with severe acute respiratory syndrome coronavirus 2 occurring several months after the primary infection reports the longest time interval between reinfection and initial infection described to date. It raises concerns on the duration of protective immunity, suggesting that it may begin to wane in patients who acquired the initial infection during the first wave of the pandemic. The potential contributing role of arterial hypertension and cardiometabolic comorbidities as risk factors for reinfection deserves investigation.

Durable antibody response one year after hospitalization for COVID-19: A longitudinal cohort study.

OBJECTIVES: Durability of the humoral immune response to SARS-CoV-2 has yet to be defined. We longitudinally evaluated during a 12-month period the antibody responses to SARS-CoV-2, and analysed predictors of antibody titres decline and seroreversion. METHODS: Prospective study conducted in a cohort of patients hospitalized for microbiologically-confirmed COVID-19. Blood and nasopharyngeal samples were sequentially obtained during hospital stay and at 1, 2, 6 and 12 months after patients' discharge for measuring anti-spike (S) and anti-nucleocapsid (N) IgG antibody levels and SARS-CoV-2 RNA, respectively. RESULTS: 80 non-vaccinated patients were analysed. At month 12 after discharge, 73 (91.2%) patients exhibited detectable S-IgG and 35 (43.8%) N-IgG antibody titres. A gradual wane was observed in S-IgG and N-IgG antibody titres. Linear regression showed that S-IgG decline was positively associated with peak antibody titres (coefficient [95% CI] 0.059 [0.05-0.067], p < 0.001), inversely with WHO severity score (coefficient [95% CI] -0.042 [-0.079/-0.004], p = 0.033), and there was a trivial positive association with age (coefficient [95% CI] 0.002 [0-0.005], p = 0.10); N-IgG decline was positively associated with peak antibody titres (coefficient [95% CI] 0.091 [0.078-0.105], p < 0.001). Logistic regression showed that seroreversion for S-IgG was inversely associated with peak S-IgG (OR 0.19; 95% CI, 0.04-0.45; p = 0.004); seroreversion for N-IgG was inversely associated with peak N-IgG (OR 0.71; 95% 0.53-0.90; p = 0.009) and positively with cycle threshold of RT-PCR (OR 1.14; 95% CI, 1.00-1.33; p = 0.062). CONCLUSION: Anti-spike IgG antibodies remain detectable one year after hospitalization for COVID-19. Higher peak antibody titres and disease severity were associated with increased durability of detectable antibodies.

Nasopharyngeal Panbio COVID-19 Antigen Performed at Point-of-Care Has a High Sensitivity in Symptomatic and Asymptomatic Patients With Higher Risk for Transmission and Older Age.

BACKGROUND: Performance of point-of-care tests in different clinical scenarios and on different samples remains undetermined. We comprehensively evaluated the performance of the nasopharyngeal Panbio COVID-19 Ag Rapid Test Device. METHODS: This is a prospective study that includes consecutive patients attending 3 primary care centers (PCCs) and an emergency department. The antigen test was performed at point-of-care in nasopharyngeal and nasal swabs and in saliva. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated with the reverse-transcription polymerase chain reaction (RT-PCR) assay as reference standard. RESULTS: Of 913 patients included, 296 (32.3%) were asymptomatic and 690 (75.6%) came from the PCC. Nasopharyngeal swabs were collected from 913 patients, nasal swabs were collected from 659 patients, and saliva was collected from 611 patients. The RT-PCR was positive in 196 (21.5%) nasopharyngeal samples (NPS). Overall, PPA (95% CI) in NPS was 60.5% (53.3-67.4), and it was lower in nasal swabs (44.7%) and saliva (23.1%). Test performance in NPS was largely dependent on the cycle threshold (Ct) in RT-PCR, with PPA of 94% for Ct ≤25 and 80% for Ct <30. In symptomatic patients, the PPA was 95% for Ct ≤25, 85% for Ct <30, and 89% for the symptom triad of fever, cough, and malaise. Performance was also dependent on age, with a PPA of 100% in symptomatic patients >50 years with Ct <25. In asymptomatic patients, the PPA was 86% for Ct <25. In all cases, NPA was 100%. CONCLUSIONS: The nasopharyngeal Panbio COVID-19 Ag test performed at point-of-care has a good sensitivity in symptomatic patients with Ct <30 and older age. The test was useful to identify asymptomatic patients with lower Ct values.

Clinical evaluation of a new molecular method for the detection of multidrug-resistant microorganisms. / Evaluación clínica de un nuevo método molecular para la detección de microorganismos multirresistentes.

INTRODUCTION: The main objective of this work is to carry out the clinical validation of the trial with the AMR Direct Flow Chip® starting from either nasal swabs, rectal swabs directly or from isolated strains to detect antibiotic resistance genes. METHODS: We developed the preclinical validation of the assay with 104 known bacterial isolates. A total of 210 nasal or rectal swab samples were analyzed. The AMR assay is based on multiplex PCR followed by reverse dot blot hybridization on DNA arrays fully automated by using the HS24 platform. The completion time of the full analysis is 3 hours. RESULTS: Both the sensitivity and specificity of the preclinical assay were 100%, with the 104 samples correctly identified. In the clinical validation, the sensitivity was 100% and the specificity was between 100% in rectal swabs and 97% in nasal swabs. CONCLUSIONS: The AMR Direct Flow Chip® is a rapid and effective assay for the detection of multidrug-resistant microorganisms from nasal and rectal swab samples.

Performance of Saliva Specimens for the Molecular Detection of SARS-CoV-2 in the Community Setting: Does Sample Collection Method Matter?

Data on the performance of saliva specimens for diagnosing coronavirus disease 2019 (COVID-19) in ambulatory patients are scarce and inconsistent. We assessed saliva-based specimens for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcriptase PCR (RT-PCR) in the community setting and compared three different collection methods. This prospective study was conducted in three primary care centers. RT-PCR was performed on paired nasopharyngeal swabs (NPS) and saliva samples collected from outpatients with a broad clinical spectrum of illness. To assess differences in collection methods, saliva specimens were obtained in a different way in each of the participating centers: supervised collection (SVC), oropharyngeal washing (OPW), and self-collection (SC). Pairs of NPS and saliva samples from 577 patients (median age, 39 years; 44% men; 42% asymptomatic) were collected and tested, and 120 (20.8%) gave positive results. The overall agreement with NPS results and kappa coefficients (κ) for saliva samples obtained by SVC, OPW, and SC were 95% (κ = 0.85), 93.4% (κ = 0.76), and 93.3% (κ = 0.76), respectively. The sensitivities (95% confidence intervals [95% CI]) of the saliva specimens ranged from 86% (72.6% to 93.7%) for SVC to 66.7% (50.4% to 80%) for SC samples. Sensitivity was higher for samples with lower cycle threshold (CT ) values. The best RT-PCR performance was observed for SVC, with sensitivities (95% CI) of 100% (85.9% to 100%) in symptomatic individuals and 88.9% (50.7% to 99.4%) in asymptomatic individuals at CT values of ≤30. We conclude that saliva is an acceptable specimen for the detection of SARS-CoV-2 in the community setting. Specimens collected under supervision perform comparably to NPS and can effectively identify individuals at higher risk of transmission under real-life conditions.

Author Correction: A nonlinear time-series analysis approach to identify thresholds in associations between population antibiotic use and rates of resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Asthma prevalence in patients with SARS-CoV-2 infection detected by RT-PCR not requiring hospitalization.

INTRODUCTION: The prevalence of asthma in patients hospitalized with SARS-CoV-2 has been studied and varies widely in the different series. However, the prevalence in SARS-infected patients not requiring hospitalization is not known. The objective of this study was to analyze the presence of asthma in a consecutive series of patients who tested positive in the RT-PCR assay for SARS-CoV-2 and did not require hospital admission. METHODS AND RESULTS: A total of 218 patients (58% of those who tested positive) did not require hospitalization; they had a median age of 45 years (IQR 34-57) and 57% were female. Six patients (2.8%) had a previous diagnosis of asthma. Only one patient developed a mild aggravation of asthma symptoms associated with SARS-CoV-2 infection. CONCLUSIONS: Few patients with asthma were infected by SARS-CoV-2, and this infection was not a significant cause of asthma exacerbation.

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain.

BACKGROUND: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened. METHODS AND FINDINGS: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region. CONCLUSIONS: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control.

A nonlinear time-series analysis approach to identify thresholds in associations between population antibiotic use and rates of resistance.

Balancing access to antibiotics with the control of antibiotic resistance is a global public health priority. At present, antibiotic stewardship is informed by a 'use it and lose it' principle, in which antibiotic use by the population is linearly related to resistance rates. However, theoretical and mathematical models suggest that use-resistance relationships are nonlinear. One explanation for this is that resistance genes are commonly associated with 'fitness costs' that impair the replication or transmissibility of the pathogen. Therefore, resistant genes and pathogens may only gain a survival advantage where antibiotic selection pressures exceed critical thresholds. These thresholds may provide quantitative targets for stewardship-optimizing the control of resistance while avoiding over-restriction of antibiotics. Here, we evaluated the generalizability of a nonlinear time-series analysis approach for identifying thresholds using historical prescribing and microbiological data from five populations in Europe. We identified minimum thresholds in temporal relationships between the use of selected antibiotics and incidence rates of carbapenem-resistant Acinetobacter baumannii (Hungary), extended-spectrum ß-lactamase-producing Escherichia coli (Spain), cefepime-resistant E. coli (Spain), gentamicin-resistant Pseudomonas aeruginosa (France) and methicillin-resistant Staphylococcus aureus (Northern Ireland) in different epidemiological phases. Using routinely generated data, our approach can identify context-specific quantitative targets for rationalizing population antibiotic use and controlling resistance. Prospective intervention studies that restrict antibiotic consumption are needed to validate these thresholds.

[Expanded-spectrum beta-lactamase-producing strains of E. coli: origin, characteristics and incidence in Southern Alicante (Spain) in the period 1999-2003]. / Cepas de Escherichia coli productoras de betalactamasas de espectro extendido: origen, características e incidencia en el sur de la provincia de Alicante en el período 1999-2003.

INTRODUCTION: In the last years, we have verified the increasing emergence of bacteria, specially Escherichia coli, that produce expanded spectrum beta-lactamases (ESBL), enzymes which confer resistance to all cephalosporins (except cephamycins) and aztreonam. These bacteria are frequently resistant also to non-beta-lactam antibiotics, a fact which poses an important clinical problem. METHODS: Descriptive study of ESBL-producing strains of E. coli isolated in all kind of specimens in two hospitals of Southern Alicante (Spain), throughout a period of 57 months (January 1999 to September 2003), paying a close attention to their origin (outpatients or admitted patients), co-resistance to non beta-lactam antibiotics and evolution of their incidence. RESULTS: Respectively, 3% and 2.25% of E. coli strains isolated in each hospital produce ESBL (3.83% and 2.85% of strains from admitted and 2.74% and 2.1% from outpatients). 30.73% and 24.58% of strains ESBL were isolated in admitted patients. We found in both hospitals much higher percentages of co-resistance to ciprofloxacin, gentamicin and trimetoprim-sulfamethoxazole in ESBL-producing strains. CONCLUSION: The percentage of ESBL-producing E. coli is high in our environment, but it is even more noteworthy its clear trend to increase. It is very remarkable the high percentage of ESBL-producing strains isolated from outpatients. Finally, we emphasize the high percentages of co-resistance to non-beta-lactam antibiotics.