Mycobacterium tuberculosis and whole genome sequencing: a practical guide and online tools available for the clinical microbiologist

Whole genome sequencing (WGS) has the potential to revolutionize the diagnosis of Mycobacterium tuberculosis (MTB) but the lack of bioinformatic expertise among clinical microbiologists is a barrier for adoption. Software products for analysis should be simple, free of charge, able to accept data directly from the sequencer (FASTQ files) and to provide the basic functionalities all-in-one. The main aim of this narrative review is to provide a practical guide for the clinical microbiologist, with little or no practical experience of WGS analysis, with a specific focus on software products tailored made for MTB analysis. With sequencing performed by an external provider, it is now feasible to implement WGS analysis in the routine clinical practice of any microbiology laboratory, with the potential to detect resistance weeks before traditional phenotypic culture methods, but the clinical microbiologist should be aware of the limitations of this approach

Prevalence and clinical correlations of genetic subtypes of Giardia lamblia in an urban setting

The clinical significance of different genetic subtypes or assemblages of Giardia lamblia is uncertain. Cases of giardiasis in south-west London between 1999 and 2005 were studied, comparing molecular-typing results with clinical and epidemiological findings from routine surveillance. We identified 819 cases, of whom 389 returned surveillance questionnaires. A subset of 267 faecal samples was submitted for typing by sequencing of the triose phosphate isomerase (tpi) and ribosomal RNA genes, and/or a separate duplex PCR of the tpi gene. Typing was successful in 199 (75%) samples by at least one of the molecular methods. Assemblage A accounted for 48 (24%) samples and Assemblage B for 145 (73%); six (3%) were mixed. Both assemblages had similar seasonality, age distribution and association with travel. Clinical features were available for 59 successfully typed cases: both assemblages caused similar illness, but Assemblage A was significantly more frequently associated with fever than Assemblage B

The complex evolution of antibiotic resistance in Mycobacterium tuberculosis.

Multidrug-resistant and extensively drug-resistant tuberculosis (TB) represent a major threat to the control of the disease worldwide. The mechanisms and pathways that result in the emergence and subsequent fixation of resistant strains of Mycobacterium tuberculosis are not fully understood and recent studies suggest that they are much more complex than initially thought. In this review, we highlight the exciting new areas of research within TB resistance that are beginning to fill these gaps in our understanding, whilst also raising new questions and providing future directions

Validation and clinical application of molecular methods for the identification of molds in tissue

Background. Invasive fungal infections due to less-common molds are an increasing problem, and accurate diagnosis is difficult.Methods. We used our previously established molecular method, which allows species identification of molds in histological tissue sections, to test sequential specimens from 56 patients with invasive fungal infections who were treated at our institution from 1982 to 2000.Results. The validity of the method was demonstrated with the establishment of a molecular diagnosis in 52 cases (93%). Confirmation of the causative organism was made in all cases in which a mold had been cultured from the tissue specimen. Less-common molds were identified in 7% of cases and appear to be an increasing problem.Conclusions. Our previously established method has proven to be of value in determining the incidence of invasive infection caused by less-common molds. Institutions should continue to pursue diagnosis of invasive fungal infections by means of tissue culture and microbiologic analysis

Self-assembled nanoparticles as multifunctional drugs for anti-microbial therapies

crosscheck: This document is CrossCheck deposited related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: The accepted version of this article will be made freely available after a 12 month embargo period history: Received 15 January 2014; Accepted 22 May 2014; Accepted Manuscript published 22 May 2014; Advance Article published 4 June 2014; Version of Record published 19 June 201

Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples.

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis

Who gets a laboratory positive diagnosis of Mycoplasma pneumoniae: A 10-year retrospective analysis

OBJECTIVE: Mycoplasma pneumoniae (M. pneumoniae) is thought to cause up to a third of community acquired pneumonias (CAP), but may be undiagnosed due to limitations with current diagnostics, and untreated given the frequent use of B-lactams to which M. pneumoniae is not susceptible. We performed a ten-year retrospective analysis to identify the typical characteristics of a patient with a laboratory positive diagnosis of M. pneumoniae. METHODS: Laboratory diagnosis of M. pneumoniae was performed using Polymerase Chain Reaction (PCR) and Serology (passive particle agglutination (PPA) and Enzyme-linked immunosorbent (EIA) assays). Data were collected on all patients tested for M. pneumoniae between 2009 and 2019. RESULTS: 19,090 PCR and 4530 serology samples were tested for M. pneumoniae with 278 positive results. The positive group had a median age of 40 years (interquartile range 30–41 years); Median C-reactive Protein (CRP) was 71 mg/L, White blood Cell Count (WBC) 7 × 10^{9}. 80% had an abnormal Chest X-ray. Intensive Care Unit (ICU) admission occurred in 4.5%; 1.3% patients died. 29% of patients were positive on both serology and PCR testing platforms. CONCLUSIONS: The characteristics reported here could be used as guidance on who is treated for M. pneumoniae. We propose that testing for M. pneumoniae needs to be performed systematically in patients with CAP; and that targeted atypical pathogen cover should be considered in preference to B-lactam mono-therapy for all patients with these characteristics

Whole genome sequencing and prediction of antimicrobial susceptibilities in non-tuberculous mycobacteria

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens commonly causing chronic, pulmonary disease which is notoriously hard to treat. Current treatment for NTM infections involves at least three active drugs (including one macrolide: clarithromycin or azithromycin) over 12 months or longer. At present there are limited phenotypic in vitro drug susceptibility testing options for NTM which are standardised globally. As seen with tuberculosis, whole genome sequencing has the potential to transform drug susceptibility testing in NTM, by utilising a genotypic approach. The Comprehensive Resistance Prediction for Tuberculosis is a database used to predict Mycobacterium tuberculosis resistance: at present there are no similar databases available to accurately predict NTM resistance. Recent studies have shown concordance between phenotypic and genotypic NTM resistance results. To benefit from the advantages of whole genome sequencing, further advances in resistance prediction need to take place, as well as there being better information on novel drug mutations and an understanding of the impact of whole genome sequencing on NTM treatment outcomes