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.

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.

Diagnóstico microbiológico de la bacteriemia y la fungemia: hemocultivos y métodos moleculares / Microbiological diagnosis of bacteraemia and fungaemia: Blood cultures and molecular methods

OBJETIVO: En esta revisión se pretende actualizar los nuevos procedimientos aplicables en el diagnóstico microbiológico de las bacteriemias y fungemias. MÉTODO: Revisión de la literatura científica. RESULTADOS Y CONCLUSIONES: Tras definir el proceso e indicar sus principios fundamentales, se revisan los principales biomarcadores utilizados en la práctica clínica. Posteriormente, se resaltan las particularidades de la fase preanalítica (recogida y transporte de las muestras) y se detallan los pasos a seguir para la identificación microbiológica por métodos clásicos, basados en el cultivo de las muestras de sangre. En el siguiente apartado, se revisan los métodos diagnósticos no basados en el cultivo, incluyendo los que detectan la presencia del genoma del microorganismo y los basados en el estudio del proteoma mediante espectrometría de masas. En el último apartado se describen los procedimientos a seguir para el estudio de la sensibilidad antibiótica, tanto por métodos fenotípicos como genotípicos

OBJETIVE: In this review we try to update the new procedures applicable in the microbiological diagnosis of bacteriemia and fungemias. METHOD: Review of scientific literature. RESULTS AND CONCLUSIONS: After defining the process and indicating its fundamental principles, the main biomarkers used in clinical practice are reviewed. Subsequently, the particularities of the pre-analytical phase (collection and transport of samples) are highlighted and the steps to follow for the microbiological identification by classical methods are detailed, based on the culture of the blood samples. In the following section, we review the diagnostic methods not culture based, including those that detect the presence of the genome of the microorganism and those based on the study of proteome by mass spectrometry. The last section describes the procedures more frecuently used for the study of antibiotic susceptibility, both by phenotypic and genotypic methods

Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

Understanding why some multidrug-resistant tuberculosis cases are not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostic assays and advance toward personalized tuberculosis treatment. Here, we combine whole-genome sequencing with single-colony phenotyping to identify a multidrug-resistant strain that had infected a patient for 9 years. Our investigation revealed the failure of rapid testing and genome-based prediction tools to identify the multidrug-resistant strain. The false-negative findings were caused by uncommon rifampicin and isoniazid resistance mutations. Although whole-genome sequencing data helped to personalize treatment, the patient developed extensively drug-resistant tuberculosis, highlighting the importance of coupling new diagnostic methods with appropriate treatment regimens.

Microbiological diagnosis of bacteraemia and fungaemia: Blood cultures and molecular methods. / Diagnóstico microbiológico de la bacteriemia y la fungemia: hemocultivos y métodos moleculares.

OBJETIVE: In this review we try to update the new procedures applicable in the microbiological diagnosis of bacteriemia and fungemias. METHOD: Review of scientific literature. RESULTS AND CONCLUSIONS: After defining the process and indicating its fundamental principles, the main biomarkers used in clinical practice are reviewed. Subsequently, the particularities of the pre-analytical phase (collection and transport of samples) are highlighted and the steps to follow for the microbiological identification by classical methods are detailed, based on the culture of the blood samples. In the following section, we review the diagnostic methods not culture based, including those that detect the presence of the genome of the microorganism and those based on the study of proteome by mass spectrometry. The last section describes the procedures more frecuently used for the study of antibiotic susceptibility, both by phenotypic and genotypic methods.

[Quality control in molecular microbiology]. / Control de calidad en microbiología molecular.

The term quality assurance (QA) refers to the quality control activities related to analytical procedures performed in the clinical microbiology laboratory. QA should include both external and internal quality assessment. Application of quality control tools in molecular microbiology assays is crucial to ensure the accuracy of results and appropriate patient management. External quality control is used for laboratory intercomparisons, detection of random and systematic errors, evaluation of the suitability of some reagents or commercial diagnostic kits, and continuing education. The External Quality Control Program of the Spanish Society of Infectious Diseases and Clinical Microbiology includes quality control procedures for molecular microbiology, as well as specific programs for quantitative determination of the viral load of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV), two highly important molecular markers in clinical settings due to their prognostic value and utility as a treatment guide. Internal quality control allows random and systematic errors to be detected through the inclusion of quality control samples in the assays performed in the laboratory, equipment monitoring, and audit. Evaluation of all molecular microbiology assays before their inclusion in the daily routine work of the laboratory is of utmost importance.