In-Depth Study of a Nosocomial Outbreak Caused by Extensively Drug-Resistant Pseudomonas aeruginosa Using Whole Genome Sequencing Coupled With a Polymerase Chain Reaction Targeting Strain-Specific Single Nucleotide Polymorphisms.

In 2013-2014, an outbreak involving 14 patients infected by an extensively drug-resistant strain of Pseudomonas aeruginosa was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy for investigating the outbreak in depth by means of molecular and genomic approaches. Pulsed-field gel electrophoresis (PFGE) was applied as a first-line approach, followed by a more refined whole genome sequencing analysis. Single nucleotide polymorphisms identified by whole genome sequencing were used to design a specific polymerase chain reaction (PCR) for screening unsuspected cases infected by the outbreak strain. Whole genome sequencing alerted us to the existence of greater genetic diversity than was initially assumed, splitting the PFGE-associated outbreak isolates into 4 groups, 2 of which represented coincidental transmission unrelated to the outbreak. A multiplex allele-specific PCR targeting outbreak-specific single nucleotide polymorphisms was applied to 290 isolates, which allowed us to identify 25 additional cases related to the outbreak during 2011-2017. Whole genome sequencing coupled with an outbreak-strain-specific PCR enabled us to markedly redefine the initial picture of the outbreak by 1) ruling out initially suspected cases, 2) defining likely independent coincidental transmission events, 3) predating the starting point of the outbreak, 4) capturing new unsuspected cases, and 5) revealing that the outbreak was still active.

Analysis of drug resistance mutations in whole blood DNA from HIV-1 infected patients by single genome and ultradeep sequencing analysis.

In HIV-1 infected patients blood CD4+ T lymphocytes could be a valuable target to analyse drug resistance mutations (DRM) selected over the course of the infection. However, detection of viral resistance mutations in cellular DNA by standard genotype resistance techniques (SGRT) is suboptimal. Whole blood DNA (wbDNA) from 12 HIV-1 infected patients on ART was studied by Single Genome Sequencing (SGS) and 8 of them also by Ultradeep pyrosequencing (UDP). Results were compared with contemporary and historical DRM detected in plasma by SGRT during follow up. All the contemporary DRM detected in plasma from the viremic patients were detected by SGS and UDP (20 from 7 patients and 4 from 5 patients respectively). Out of the 67 historical DRM detected in plasma and no longer present at the time of testing, 38 (57%) were detected by SGS in 12 patients and 27 out of 46 (59%) by UDP in 8 patients. Additional DRM never reported in plasma by SGRT were detected by SGS (12 from 8 patients) and UDP (10 from 8 patients). Analysis of wbDNA from HIV-1 infected patients by SGS and UDP provides proof of concept of the value of blood DNA to investigate current and archived DRM in HIV-1 infected patients on ART.

In-Depth Characterization and Functional Analysis of Clonal Variants in a Mycobacterium tuberculosis Strain Prone to Microevolution.

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. Four different clonal variants were identified from independent intrapatient evolutionary branches. One of the single-nucleotide polymorphisms in the variants mapped in mce3R, which encodes a repressor of an operon involved in virulence, and affected expression of the operon. Competitive in vivo and in vitro co-infection assays revealed higher infective efficiency for one of the clonal variants. A new clonal variant, which had not been observed in the clinical isolates, emerged in the infection assays and showed higher fitness than its parental strain. The analysis of other patients involved in the same transmission cluster revealed new clonal variants acquired through novel evolutionary routes, indicating a high tendency toward microevolution in some strains that is not host-dependent. Our study highlights the need for integration of various approaches to advance our knowledge of the role and significance of microevolution in tuberculosis.

Lack of the detection of XMRV or polytropic MLV-related sequences in blood cells from HIV-1-infected patients in Spain.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)-related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus. METHODS: Forty HIV-1-infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination. RESULTS: No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed. CONCLUSIONS: XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.