Background: Metagenomic sequencing is frequently claimed to have the potential to revolutionise microbiology through rapid species identification and antimicrobial resistance (AMR) prediction. We assess progress towards this. Methods: We perform a systematic review and meta-analysis of all published literature on culture-independent metagenomic sequencing for pathogen-agnostic infectious disease diagnostics to August 12, 2020. Methodologic bias and applicability were assessed using QUADAS-2. (PROSPERO CRD42020163777) Results: A total of 2023 clinical samples from 13/21 eligible diagnostic test accuracy studies were included in the meta-analysis. Reference standards were culture, molecular testing, clinical decision or a composite measure. Sensitivity and specificity in the most widely investigated sample types were 90%(78-96%) and 86%(45-98%) for blood, 75%(95%CI, 54-89%) and 96%(72-100%) for CSF, and 84%(79-88%) and 67%(38-87%) for orthopaedic samples respectively. We identified limited use of controls, especially negative controls which were used in only 62%(13/21) studies. AMR prediction and comparison to phenotypic results was undertaken in four studies: categorical agreement was 88%(80%-97%), very major and major error rates were 24%(8-40%) and 5%(0-12%) respectively. Better human DNA depletion methods are required: a median 91%(IQR 82-98%)[range 76-98%] of sequences were classified as human. The median(IQR)[range] time from sample to result was 29(24-94)[4-144] hours. The reported consumables cost per sample ranged from $130-$685. Conclusions: There is scope for improving the quality of reporting in clinical metagenomic studies. Although our results are limited by the heterogeneity displayed, our results reflect a promising outlook for clinical metagenomics. Methodological improvements, and convergence around protocols and best practises may improve performance in future.
The detection of herpes, chancroid, and syphilis in genital ulcers is done by PCR. This is not so for lymphogranuloma venereum (LGV). We report on the use of a PCR with digestion that differentiates the LGV biovar from the trachoma biovar. Our findings suggest that the clinical description of LGV in current textbooks is incomplete.
Culture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and taxonomic classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of the ten common bloodstream pathogens and six frequent blood-culture contaminants (n=1568, only 68 genomes were available for Micrococcus luteus ). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken and Centrifuge, utilizing mini (8 GB) and standard (30–50 GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 97.8% (IQR 92.7:99.0) [range 5:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 86.4% (IQR 50.5:93.7) [range 4.3:100]. Classification performance varied by species, with Escherichia coli being more challenging to classify correctly (probability of reads being assigned to the correct species: 56.1–96.0%, varying by tool used). Human read misclassification was negligible. By filtering out shorter Nanopore reads we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. To account for ‘bioinformatic contamination’ we present a contamination catalogue that can be used in metagenomic pipelines to ensure accurate results that can support clinical decision making.
Culture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of ten common bloodstream pathogens and six frequent blood culture contaminants (n=1600). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken, and Centrifuge, utilising mini (8GB) and standard (30-50GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 98.46% (IQR 93.0:99.3) [range 57.1:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 79.3% (IQR 39.1:88.8) [range 11.2:100]. Classification performance varied by species, with E. coli being more challenging to classify correctly (59.4% to 96.4% reads with correct species, varying by tool used). By filtering out shorter Nanopore reads (<3500bp) we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. This “bioinformatic contamination” should be accounted for in metagenomic pipelines to ensure accurate results that can support clinical decision making.
Background: A summary of factors associated with recurrent tuberculosis (TB) in the African HIV-infected population is lacking. We performed a systematic review to address this. Methods: We performed a literature search within PubMed and The WHO Global Library with specific inclusion and exclusion criteria to identify manuscripts emanating from the African continent which potentially described factors associated with recurrent TB in persons living with HIV. Results: The literature search yielded 52 unique manuscripts, of which only 4 manuscripts were included in the final systematic review following application of the inclusion and exclusion criteria. Baseline CD4 count, baseline HIV viral load, a positive tuberculin skin test, prior active TB disease, cutaneous hypersensitivity reaction to treatment, having < 3 lung zones affected by prior TB disease, and anaemia were associated with recurrent TB in HIV-infected individuals, whilst age and antiretroviral status were not. Conclusion:The lack of studies describing recurrent TB in Africa which stratify results by HIV-status is a hindrance to understanding risk factors for recurrent TB in this population. This might be overcome by implementing guidelines related to the publishing of data from observational studies in peer-reviewed medical journals reporting recurrent TB in populations with a high-burden of HIV infection.
Background: A contemporary summary describing the impact of an HIV-positive status on short term perioperative mortality is lacking. Objective: To collate and summarise published data related to short term perioperative mortality from studies comparing HIVpositive and HIV-negative patient groups. Method: We conducted a systematic review of the published literature by performing structured searches of two medical literature databases. Pre-defined inclusion/exclusion criteria were used to identify potentially relevant manuscripts. Further screening of the reference lists of eligible manuscripts, as well as a prior systematic review was also performed to identify any additional manuscripts that may have been relevant. Data retrieved from eligible manuscripts included, amongst other variables: study and population descriptions, surgical category (cardiac or noncardiac surgery), as well the incidence of short term perioperative mortality. Crude odds ratios were calculated for each eligible manuscript to describe the association between HIV status and short term perioperative mortality. Results: Our systematic review consisted of 12 manuscripts describing 12 studies. The majority of manuscripts described studies conducted in countries with a low burden of HIV infection. Most manuscripts described findings from a noncardiac surgery setting. Crude associations between an HIV-positive status and a higher odds of short term perioperative mortality were noted for data from 2 of the 12 manuscripts, while the association was unclear in the remaining 10 manuscripts. Conclusion: Evidence supporting a higher odds of short term perioperative mortality in patients with an HIV-positive status is unconvincing. Further research is required to adequately investigate this.
Metagenomics has the potential to revolutionise infectious diseases diagnostics, from rapid species and antimicrobial resistance prediction, to finding unrecognised and sometimes untreated infections. Our aim was to summarise all literature on culture-independent metagenomic sequencing to describe the accuracy of species and antimicrobial resistance prediction and, describe the challenges and progress in the field. We conducted a systematic review with meta-analysis from eligible studies retrieved from PubMed, Google Scholar and bioRxiv and, assessed risk of bias and quality using the QUADAS-2 tool. This study is registered with PROSPERO, number CRD42020163777. We identified 36 studies, 22 of which used a species-agnostic approach to identify all possible pathogens. In these studies, the overall sensitivity and specificity of pathogen species detection were 88% (95%CI 81-92%) and 86% (95%CI 70-94%) respectively. Antimicrobial resistance prediction and comparison to phenotypic results was undertaken in six studies. Categorical agreement was 83% (95%CI 68-92%), very major (prediction sensitive, phenotype resistant) and major error (prediction resistant, phenotype sensitive) rates were 9% (95%CI 2-27%) and 1% (95%CI 0-20%) respectively. We report limited use of negative controls in studies 61% (22/36) which contribute to a major challenge of discriminating true pathogens from contamination, where there is no convergence on methodology. More efficient human DNA depletion methods are required as a median of 79% (IQR 62-96) [Range 7-98] of sequences were classified as human despite laboratory depletion techniques. The median time from sample to result was 23.5 hours (7-31) [4-144], with sequencing time accounting 10 hours (4.8-16) [1-16]. The average reported consumables cost per sample ranged from $128 to $685. The science and regulatory environment are rapidly developing, and its role as a routine test or test of last resort still needs to be determined, however it is likely that clinical metagenomics will be an increasing part of the clinician′s armamentarium to diagnose infectious diseases in the near future.
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